N = 9399) were 6.7  times more likely to die than the general population, but this did not vary by migrant status (P = .15) or region-of-origin (P = .31). This mortality gap was most pronounced for suicide , but persisted for deaths from other external  and natural causes . Non-refugee  and refugee migrants  were more likely to receive inpatient care at first diagnosis. No differences in in-patient LOS at first diagnosis were observed by migrant status. Sub-Saharan African migrants with psychotic disorder were readmitted more quickly than their Swedish-born counterparts . Our findings highlight the need to understand the drivers of disparities in psychosis treatment and the mortality gap experienced by all people with disorder, irrespective of migrant status or region-of-origin.It is unclear whether inequalities in mental healthcare and mortality following the onset of psychosis exist by migrant status and region-of-origin. We investigated whether (1) mortality (including by major causes of death); (2) first admission type (inpatient or outpatient); (3) in-patient length of stay (LOS) at first diagnosis for psychotic disorder presentation, and; (4) time-to-readmission for psychotic disorder differed for refugees, non-refugee migrants, and by region-of-origin. We established a cohort of 1 335 192 people born 1984–1997 and living in Sweden from January 1, 1998, followed from their 14th birthday or arrival to Sweden, until death, emigration, or December 31, 2016. People with ICD-10 psychotic disorder (F20–33;  For example, although more than half of those who experience a first episode of psychosis (FEP) meet criteria for clinical remission within a year of commencing treatment, recent evidence indicates that nearly 80% of patients experience relapse thereafter.9–11Psychotic disorders affect approximately 3.5% of the population,12 This risk may be even more pronounced in some groups, including people from visible minority backgrounds,12 and refugees13 who were 3 times more likely to be diagnosed with non-affective psychotic disorder than the native Swedish-born population. In the United Kingdom, there is evidence that people from Black, Asian, and minority ethnic backgrounds with FEP also experience greater excess mortality than the general population.15 There is also strong evidence from the United Kingdom that people with FEP from some ethnic minority backgrounds—most notably from Black ethnic backgrounds—are more likely to be compulsorily admitted or readmitted for care for any psychiatric disorder.16 There is more mixed evidence about ethnic disparities in length of stay (LOS) for inpatient stays following FEP in the United Kingdom. Two studies suggest that several ethnic groups,17 including people of Black ethnic origin18 experienced longer LOS than White participants, and that LOS was longer for people diagnosed with psychosis than other conditions.17 By contrast, another study reported a reduction in inpatient stays in Black Caribbean compared with White FEP participants receiving intensive community treatment.19 There is also some evidence that people of Black and Asian ethnic origins in the United Kingdom were more and less likely, respectively, to be readmitted following FEP than their White counterparts.20Given strong evidence that migrants and their children are on average 2 times more likely to develop a psychotic disorder than the majority population these inequalities may disproportionately affect migrant and minority ethnic communities.21 For example, no previous studies have examined whether the mortality gap between people with FEP and the general population varies by migrant status, although migrants in general population samples appear to have lower mortality rates than native-born populations,23 and migrants with FEP appear less likely to die by suicide,21 as found in the general population.24 Recent evidence from Sweden has found that several migrant groups were at elevated risk of compulsory admission compared with Swedish-born individuals,25 supporting UK findings. With respect to readmission, data from New Zealand supports higher readmission rates following a psychiatric diagnosis,26 including FEP,27 for people of Maori ethnicity compared with the White majority group, while another Canadian study found no evidence that ethnicity was a predictor of admission after a FEP diagnosis.28 In Switzerland, one study found that immigrants had lower readmission rates than their Swiss-born counterparts,29 but another recent study from the same country suggested higher relapse rates and worse symptomatology amongst migrants who had also experienced adversity.30 In the United States, another study reported that African-American participants with psychotic disorders had shorter LOS than White participants, while Asian and Native American participants experienced longer LOS.31 No differences in LOS by migrant status were reported in a study in Italy.32Outside of the United Kingdom, these issues have received less attention, with more mixed evidence available.To our knowledge, no study has examined disparities in multiple outcomes following FEP—including mortality, type of admission (inpatient or outpatient), LOS or readmission risk—by migrant status in a single study. Understanding these issues would provide vital information for service planners and public health to reduce potential inequalities in psychosis care. We investigated these issues in a cohort of people with a first diagnosis of non-organic psychotic disorder in Sweden, using national register data. Sweden provides a highly suitable setting to investigate these issues. It has a universal healthcare system, including for psychiatric care, free at the point of access for all residents, including refugees. As an European Union (EU) member state, citizens of all EU countries are entitled to work and live in Sweden. In addition, Sweden provides asylum to a large number of refugees and their families from many regions, particularly East Africa, Iraq, Iran, Afghanistan, and countries which were part of the former Yugoslavia . Before 2016, Sweden received more refugees than any other EU county on a per capita basis. We hypothesized that compared with the Swedish-born population with a psychotic disorder, refugee and non-refugee migrants with a psychotic disorder would be more likely to (1) receive inpatient hospital care at first presentation; (2) have longer inpatient care at first admission; (3) be readmitted more quickly after their first recorded diagnosis; and (4) face similar mortality gaps to their Swedish-born counterparts. Consistent with evidence elsewhere on ethnic disparities in mental healthcare, we hypothesized that where these inequalities existed, they would be greater for refugee and non-refugee migrants from regions-of-origin where participants were more likely to be of visible minority status in Sweden .33 Migrants who arrived before 1998 were excluded because data on refugee status was not routinely available before this time. We also excluded undocumented or temporary migrants, and people born in Sweden to at least one foreign-born parent from this study.Our initial sample was extracted from Psychiatry Sweden, a large, longitudinal database of several linked national Swedish registers containing anonymized data on all people in Sweden granted permanent residency. We used the Register of the Total Population (RTP) to define an initial cohort of people, born between 1984 and 1997, alive and living in Sweden on their 14th birthday, or date of arrival in Sweden, if later. Our cohort was restricted to people born in Sweden to 2 Swedish-born parents, or refugee and non-refugee migrants (born overseas) who arrived in Sweden from January 1, 1998, from regions of origin  with greater than 1000 refugees in the RTP to ensure valid comparisons , consistWe included 4 outcomes in our study: (1) all-cause mortality as recorded in the Causes of Death Register for people who did and did not receive a first diagnosis of International Classification of Diseases (10th revision) (ICD-10) non-affective or affective psychotic disorder (F20–33) recorded in the National Patient Register (NPR). In post hoc analyses, we also examined these mortality gaps for major cause-specific deaths relevant to this cohort, recorded as the underlying causes of death in the register, including (a) deaths by suicide (X60–84) and undetermined intent (Y10–34), (b) deaths due to other external causes , and (c) natural causes (causes of death defined according to ICD-10 Chapters 1–18). In a cohort restricted to people diagnosed with psychotic disorder, we also included the following outcomes, (2) admission type at first presentation (inpatient or outpatient) as recorded in the NPR, (3) time-to-readmission for psychotic disorder, and (4) for participants admitted for in-patient care at first diagnosis, LOS in days.For all-cause and cause-specific mortality, we followed the whole sample from their 14th birthday or date of immigration if later, until date of death, emigration or December 31, 2016, whichever was sooner. For first admission type, if a participant had both in patient and outpatient records for the same day, we recorded this as an inpatient contact. For readmission, we followed participants from date of first diagnosis until a second contact in the NPR for psychotic disorder, emigration, death, or December 31, 2016, whichever was sooner. We defined a second contact  as a second recorded diagnosis of psychotic disorder in the NPR more than 28 days after the index diagnosis, to minimize the possibility that a second diagnostic date recorded in the NPR was part of a participant’s treatment for their first diagnosis. This cut-off was chosen based on local clinical and register expertise . Finally, to estimate inpatient LOS at first diagnosis, we estimated the days between the inpatient date and discharge date recorded in the NPR.For mortality as an outcome, our primary exposure was a diagnosis of psychotic disorder. For other outcomes around psychiatric care use, our primary exposure was migrant status, defined as people (1) born in Sweden to 2 Swedish-born parents ; (2) people granted asylum under Swedish law and meeting the UNHCR definition of refugee status due to threat of persecution, conflict or other humanitarian crises  (henceforth refugee migrants); and (3) people born outside of Sweden and permanently settled in Sweden without refugee status  from one of the regions-of-origin stated above. As a secondary exposure, we classified participants by region-of-origin, as defined above and in We controlled for sex, age, disposable income and urban residency at cohort entry . For mortality and readmission as outcomes, age was treated as time-varying covariate in survival analyses (see below). For other outcomes, age group at diagnosis of psychotic disorder was used. For both definitions of age, we categorized age into the following groups .34 We estimated population density from the RTP in each year, divided by the SAMS area (in square kilometers) to give the total population density (people per square kilometer [ppskm]), which we treated as a continuous variable transformed onto the natural logarithm scale given strong positive skew. For all outcomes restricted to people diagnosed with a psychotic disorder , we also controlled for diagnosis (ICD-10 non-affective [F20–29] vs affective [F30–33] psychotic disorder).Individualized disposable income was estimated from data in the longitudinal integration database for health insurance and labor market studies (LISA) register. Individualized disposable income was based on total annual family income, including benefits, pensions and salary/wages, weighted for household size and composition . The earliest age at which this was recorded in the LISA register was 16 years, or on arrival in Sweden if later. We classified disposable income into quintiles for the relevant calendar year, relative to the total population in Sweden, to control for inflation. Urban residency was measured as population density at age 14 years old or immigration to Sweden, if later, based on each individual’s Small Area Marketing Statistics (SAMS) area of residence. SAMS are small area geographical units created by Statistics Sweden of 1000–2000, based on election districts in more rural areas, and by dividing cities into socioeconomically homogenous neighborhoods.First, for the whole cohort, we presented descriptive statistics including crude all-cause mortality rates by migrant status, region-of-origin and diagnosis of psychotic disorder. Second, we fitted Competing Risks Regression (CRR) models to estimate unadjusted and adjusted sub-hazard ratios (sHRs) and 95% confidence interval (95%CIs) to estimate the all-cause mortality gap between the general population and participants diagnosed with psychotic disorder. Third, we tested whether this mortality gap differed by migrant status or region-of-origin, tested via likelihood ratio tests (LRTs) of models with and without the relevant interaction term(s). We repeated these analyses for cause-specific mortality gaps for people with psychotic disorder, and considered whether these differed by migrant status. We fitted cause-specific models using less computationally-intensive Cox proportional hazards models instead of CRR, as evidence from our all-cause mortality analyses suggested the 2 gave comparable point estimates of risk .We modeled time-to-readmission in the subsample of the cohort diagnosed with psychotic disorder during the follow-up period. We repeated the analytical procedure for mortality, including use of CRR, which we considered to be more appropriate than more conventional Cox proportional hazards survival analysis for both mortality and readmission outcomes, since emigration may have been a plausible competing risk for each. We analyzed the association between admission type at first diagnosis of psychotic disorder (inpatient vs outpatient) and migrant status or region-of-origin using logistic regression, and LOS as an outcome (restricted to the in-patient sample) using linear regression.a priori. We restricted analyses to complete case samples, with missingness explored descriptively. Finally, because migrant status and region-of-origin had the same reference category  we could not mutually adjust our models for both variables; to ascertain which exposure fitted the data better for each outcome, we inspected Akaike’s Information Criterion (AIC) scores in the final multivariable model fitted with each exposure, where lower score indicated better model fit having penalized for model complexity. All analyses were performed using Stata, version 15.For all outcomes, we conducted univariable and multivariable analyses, treating all confounders as This study was approved by the Stockholm Regional Ethical Review Board (2010/1185-31/5).P < .001) for refugee and non-refugee migrants. The sample also included 9399 people diagnosed for the first time with a psychotic disorder during follow-up (0.7%), which varied by migrant status  and was highest for refugees. A higher proportion of non-refugee (52.9%) and refugee (54.2%) migrants were admitted as in-patients for their first diagnosis of psychotic disorder compared with the Swedish-born population , but we observed no differences in median LOS  or proportion of participants readmitted  by migrant status. Participants from Sub-Saharan African made up the lowest proportion of non-refugee migrants (11.6%), but the second-highest proportion of refugee migrants (36.8%), while participants from the Middle East made up the highest proportion of both non-refugee (32.8%) and refugee (37.6%) migrants. Refugee migrants were more likely to be men (57.7%) than in the Swedish-born (51.3%) or non-refugee migrant (49.4%) samples (P < .001), and a higher proportion of non-refugee (93.5%) and refugee (97.4%) migrants belonged to the lowest income quintile group compared with Swedish-born participants . Median levels of population density were similar for Swedish-born and refugee participants, but notably higher for non-refugee migrants  and 24 117 refugees (1.8%) . Over thThe crude mortality rate was higher in people diagnosed with non-affective psychosis  than in the remainder of the population  and no evidence of such interaction by region-of-origin (LRT P = .31). These findings were in partial contrast to the results of the same models run using Cox proportional hazards modeling, where the presence of effect modification between psychotic disorder and migrant status on mortality would have been interpreted as conventionally meeting statistical significance  for people diagnosed with psychotic disorder than the remainder of the population, after adjusting for current age, sex, income quintile, and population density . Althoug2P-value for cause of death by migrant status on 4 degrees of freedom: 19.4(4); P = .001). People diagnosed with psychotic disorder were more likely to die from each specific cause than the population at-risk, but adjusted hazard ratio [aHRs] varied from 2.3 (95% CI: 1.6–3.4) for natural causes to 5.1  for other external causes and 12.2  for deaths by suicide or of undetermined intent. There was no statistically robust evidence that these patterns differed by migrant status , other external causes (35.5%) and natural causes (32.8%), although the former outcome was more common amongst the Swedish-born group (32.2%) than non-refugee (24.3%) and refugee (25.5%) migrants, who were more likely to die from natural causes  and refugee  migrants were more likely to be admitted as in-patients at first diagnosis of psychotic disorder, after adjustment for confounders . FurtherP = .95; P = .07).For in-patient LOS and time-to-readmission analyses, we excluded 95 participants with inconsistent inpatient dates, including a second diagnosis or death date recorded before discharge for the index diagnosis. No differences in the excluded sample were found by migrant status P = .95; . No cleaP = .04), particularly those from Sub-Saharan Africa . After further adjustment for admission type and in-patient LOS at first diagnosis, there was no longer any statistically significant evidence for shorter time-to-readmission for non-refugee migrants , though such an effect remained evident in migrants from Sub-Saharan Africa . These patterns were similar when fitted under a Cox proportional hazards model  with some evidence of shorter time-to-readmission for non-refugee migrants , we observed a large (over 6-fold) mortality gap between people diagnosed with a psychotic disorder and the remainder of the population up to 19 years after first diagnosis. This gap was evident for all broad causes of death, and most pronounced for death by suicide or of undetermined intent. Despite some variation in point estimates, there was no formal evidence that this mortality gap varied by migrant status or region-of-origin, though was present for nearly all groups save migrants of Sub-Saharan African origin. Non-refugee and refugee migrants were 40% more likely to be admitted as inpatients at first diagnosis for psychotic disorder, consistent with our earlier work that they were also more likely to be compulsorily detained. Further inspection suggested risk of inpatient admission was greatest for people from Sub-Saharan Africa and Asia in our cohort. Fewer differences in in-patient LOS at first hospitalization for psychotic disorder were observed; the average length of inpatient stay in our cohort was around 12 days. Time-to-readmission for psychotic disorder was shorter for migrants from Sub-Saharan Africa, not explained by confounding factors, including admission type or length of inpatient stay at first diagnosis.15 we identified a persistent and gross mortality gap between people with and without psychotic disorder in Sweden. This mortality gap was detectable across all groups, irrespective of migrant status or region-of-origin, consistent with findings from 2 studies in South London which found the mortality gap for people with FEP was similar across different ethnic groups.15 To our knowledge, this is the first study to inspect the mortality gap in refugees with psychotic disorder; although we did not detect statistically significant effect modification between migrant status and psychosis on all-cause or cause-specific mortality, refugees had the largest point estimate for the all-cause mortality gap (over 14 times greater for refugees with psychosis than refugees without psychosis), as well as for deaths by suicide/undetermined intent and natural causes. Larger studies are required to examine this potential disparity, but more immediately, clinicians working in refugee populations with psychotic disorders should be aware of the particularly high crude mortality rates experienced by this group (303.5 deaths per 100 000 people per year). While we have previously shown that migrants are at lower risk of death by suicide in the general population,24 this pattern differs for non-refugee and refugee migrants diagnosed with a psychotic disorder, who were, respectively over 10 and 32 times more likely to die by suicide/undetermined intent than their non-psychotic counterparts.Consistent with previous evidence,12 including higher first admission rates to hospital where this is the primary mode of care.35 Few studies have directly compared whether migrants are more or less likely to receive inpatient vs outpatient care during treatment for their first diagnosis of psychotic disorder. We extended this research to demonstrate that both non-refugee and refugee migrants were around 40% more likely to receive an inpatient admission at first diagnosis compared with their Swedish-born counterparts. Similarly to the United Kingdom,16 this risk was greatest for people from likely visible minority migrant backgrounds from Sub-Saharan Africa and Asia. Several factors may explain this finding, including the increased likelihood of compulsory admission for psychiatric care, including FEP, for people from Black, Asian and other ethnic minority backgrounds, which has been strongly and consistently observed in Sweden,25 the United Kingdom ,16 and elsewhere,36 and would typically be accompanied by inpatient rather than outpatient treatment. Greater likelihood of inpatient admission may also be explained by a longer duration of untreated psychosis [DUP] observed in some migrant groups,38 potentially delaying treatment and resulting in more severe FEP requiring hospitalization. Several factors may delay treatment-seeking in migrant groups, including unfamiliarity of the legal entitlement to healthcare in Sweden, or other cultural or structural barriers in accessing timely treatment, including language barriers, multiple complex health needs requiring treatment or fear of legal repercussions for refugees.40 We suggest that these findings will generalize to other settings where non-refugee and refugee migrants may be at both greater risk of psychosis,41 and face differential outcomes in their treatment pathways.42Previous research strongly supports higher rates of psychotic disorder amongst migrants and their children,We also observed tentative evidence, in need of replication in larger samples, that refugees experienced a 2-week longer in-patient admission at first diagnosis for psychosis than their Swedish-born counterparts. This may highlight greater additional needs in this group, and suggests that clinical services in settings with higher refugee populations will require appropriate tailoring to ensure equitable provision of effective and timely care. Unlike studies in the United Kingdom, we found no evidence of longer inpatient stays for people from likely Black backgrounds , or from any other migrant background, though imprecision around our estimates suggests our ability to detect these effects may have been limited by sample size.44 including clozapine45 to manage psychotic symptoms, while lower rates of antipsychotic medication adherence have been observed for some ethnic minority groups.47We found non-refugee migrants, but not refugees, were readmitted more quickly for psychotic disorder than the Swedish-born group over the follow-up period. This suggests further work is needed to unpack these differential findings, which were not accounted for after adjustment for a priori confounders or type of admission or length of in-patient stay. Refugees may receive additional welfare support by virtue of their legal status compared with non-refugee migrants which protects against readmission, or alternatively may be less likely to re-engage with services for some of the reasons outlined above. Previous studies have identified other inequalities in mental healthcare offered to migrants and refugees, which need to be explored in this context. For example, migrant and ethnic minority groups experiencing psychotic disorder are less likely to be offered newer second-generation antipsychotics,49 The register has achieved 100% coverage of all inpatient settings since 1987, and outpatient settings since 2006. We used CRR appropriate for 2 outcomes  as emigration (for both) and death (for time-to-readmission) may have been legitimate competing events; indeed, fitting mortality data using Cox proportional hazards models may have resulted in erroneous interpretation of the results  pathways to and through care, perhaps culminating in longer inpatient stays for refugees and quicker readmission for migrants of Sub-Saharan African origin. These findings highlight the need to examine and remove barriers to equitable service provision, including the need to design culturally sensitive, early detection and intervention services that offer accessible, destigmatized, and timely treatment for all residents in Sweden irrespective of migrant status or region-of-origin.sgab009_suppl_Supplementary_MaterialsClick here for additional data file.


Background: Microcirculation is essential for skin homeostasis and repair. A variety of growth factors have been identified as important regulators of wound healing. However, direct observation and longitudinal monitoring of skin remodeling in an unperturbed in vivo environment remains challenging.Methods: We report on non-invasive longitudinal imaging of the wound healing process in transgenic mice overexpressing vascular endothelial growth factor A (VEGF-A) in keratinocytes by means of large-scale optoacoustic microscopy (LSOM). This rapid, label-free, high throughput intravital microscopy method averts the use of dorsal skin-fold chambers, allowing for fully non-invasive repeated imaging of intact wounds with capillary resolution over field-of-view spanning several centimeters.Results: We observed VEGF-driven enhancement of dermal vascularization in ears, dorsal skin and healing wounds and quantified the hemoglobin content, fill fraction, vessel diameter and tortuosity. The in vivo findings were further corroborated by detailed side-by-side classical histological whole-mount vascular stainings and pan-endothelial CD31 immunofluorescence.Conclusion: The new approach is suitable for supplementing or replacing the cumbersome histological procedures in a broad range of skin regeneration and tissue engineering applications. Skin is the first line of defense against exogenous insults like allergens, irritants and pathogens  ex vivo, thus preventing observations of longitudinal dynamics in the same animal.Since VEGF-A target proteins and specific endothelial markers can be easily identified in tissue sections, histology and immunofluorescence stainings constitute the mainstay approach for studying effects of growth factors that control angiogenesis. However, evaluation of classical histology data is often afflicted with non-standardized procedures for tissue handling, fixation and staining, quantification challenges, and human errors in vivo. However, the minuscule field of view (FOV) Fluorescence microscopy Here, we examine the use of optical-resolution, label-free, large-scale optoacoustic microscopy (LSOM) in vivo imaging, without any prior invasive surgical procedure Materials and Methods for more details on the LSOM and AVSA methods.The label-free LSOM used for non-invasive imaging of microvascular networks in the mouse ear skin and non-injured/wounded dorsal skin is schematically displayed in Figure in vivo overview images of the whole ear along with the zoom-ins around the edges  was significantly higher (p << 0.001) than in WT mice . Similarly, the fill fraction was significantly higher  in VEGF-A mice  compared to WT mice . On the other hand, differences in the vessel diameter  and vessel tortuosity  were less pronounced.Four parameters, including Hb content, fill fraction, vessel diameter and tortuosity were analyzed by AVSA in both WT and VEGF-A mice Figure C. The Hbin vivo LSOM data, showing a higher fill fraction in VEGF-A mice , and no significant difference in tortuosity  between mice of both genotypes. The vessel diameter in WT mice was larger than in VEGF-A mice in edge regions , which is contradictory to the LSOM data (1.14-fold larger in VEGF-A mice). At base regions, there was no significant difference between WT and VEGF-A mice in both the fill fraction (p = 0.97) and vessel diameter (p = 0.30).The same vessel parameters (except Hb content) were also evaluated for whole-mount ear imaging using the same vessel analysis pipeline (AVSA) Figure D. Resulti.e., blood vessel area) and total vessel number in all locations , which is consistent with the LSOM data. A detailed statistical comparison  of the results obtained with the three techniques is shown in Table Statistical analysis of histological data from ear sections was also performed Figure B-C. CD313 FOV, 20 µm lateral step size) along with smaller dorsal region of interest  at finer 5 µm step size were recorded  was performed to quantitatively compare the difference between WT and VEGF-A mice  and vessel number  in the skin of VEGF-A versus WT mice Figure D. A detai.e., tortuous capillaries), as shown in the zoom-in LSOM images. The GT areas were also clearly observed in the photographs of mice of both genotypes. Noticeably, the distribution of dense microvessels was again significantly higher in VEGF-A mice . Progressive wound healing/closure was monitored by LSOM at days 10 and 15 post-wounding  and fill fraction  in the wounds of mice of both genotypes showed the highest values at day 10, while VEGF-A mice manifested elevated values in both metrics  were observed from day 5 to day 10 in the same VEGF-A mouse, while no significant change was observed in the WT mouse  areas with LSOM could be achieved rapidly and smoothly Based on the optical absorption curve of hemoglobin, the 532 nm wavelength is ideal for generating strong optoacoustic responses from blood e.g., when a continuous vessel is broken into smaller vessels as false branch points are detected. This issue can potentially be mitigated by processing the images with enough smoothing/filtering during the contrast enhancement procedure or selecting a binarization method with an adjustable sensitivity , so that false branches are not detected. Alternatively, images from individual cross-sections can be processed so the superficial and deeper vessels are segmented separately, thus averting the issue of small vessels crossing larger underlying vessels in the same image The application of the currently proposed AVSA algorithm may result in the skeleton of vessels becoming disconnected, Whole-mount ear imaging in mice of both genotypes was conducted and evaluated by using the same AVSA to reduce any potential bias on histological analysis, thus facilitating LSOM-based analysis of the vascular metrics. The discrepancy between statistical analysis on the LSOM datasets and whole-mount histological images pertaining vessel diameter in the edge regions  can partially be attributed to the different method of volumetric data acquisition from an intact object versus tissue sections with an additional major source of mismatch being the inferior lateral resolution of LSOM. In order to effectively match the curved surface of the mouse head, we had to opt for a large (> 1 mm) depth-of-focus, thus compromising on a relatively large 7.5 µm light spot size. In comparison, the < 2 µm resolution of the whole-mount staining reveals smaller microvascular structures in the ear. Depending on the application and desired FOV, the lateral resolution of LSOM can be further improved by increasing the numerical aperture of the focusing lens, at the expense of diminished depth of focus and/or imaged area. Additionally, CD31-stained whole-mount imaging detects both blood and lymphatic vessels, which may lead to another source of discrepancy. Note that due to the discontinuity of stained vasculature, large diameter vessels were erroneously recognized as many tiny vessels by the AVSA algorithm. Thus, the tortuosity accuracy could be further improved by attempting a more homogenous vessel staining.Hair regrowth after depilation in both WT and VEGF-A mice adversely affected the LSOM imaging quality in the wound healing experiments. This compromised the optical focusing capabilities due to scattering and acoustic coupling issues, further diminishing the signal-to-noise ratio and effective imaging depth. In the future, the skin hair growth cycle needs to be carefully monitored for rendering consistent LSOM images throughout the wound healing process. Alternatively, SKH1 hairless mice could be used for long-term skin vasculature monitoring.Direct physical contact of coupling medium with the skin wound may lead to an increased risk of infection. Indeed, non-contact imaging techniques, such as optical coherence angiography, largely decrease the risk of infection for wound monitoring. Proof-of-concepts of such non-contact studies have recently been reported i.e., top views. While LSOM images generally provide a more comprehensive undisrupted view of the vascular network, the inferior axial resolution, which is limited by the available detection bandwidth of the ultrasound transducer, compromises the depth resolving capacity of the method. Thus, optical microscopy could be used to complement the corresponding high-resolution information along the depth direction.It should be also noted that classical histology of dorsal skin and wounds is based on side-view tissue sections, whereas the LSOM images represent planar projections of volumetric data along the depth dimension, Spectroscopic optoacoustic imaging techniques employing several excitation wavelengths are generally capable of providing oxygen saturation readings to assess the relation between angiogenesis and oxygen metabolism In general, optoacoustic methods can be employed for skin research in both preclinical and clinical settings to diagnose various diseases such as psoriasis, scleroderma, and skin cancers i.e., the blind-ended lymphatic vessels/capillaries, may further help to optimize therapeutic interventions in lymphatic disorders On the other hand, the lymphatic system, present in most organs of the body, is linked to the development of many different pathologies, such as lymphedema and chronic inflammation in vivo. The epi-illumination design of LSOM could be further employed for examinations of ex vivo skin samples, and even monitoring of skull/brain regions in murine models in vivo evaluation to replace or supplement the cumbersome ex vivo histological procedures, thus demonstrating its broad potential for skin research and interrogation of diseases. Future work will focus on non-invasive assessment of therapeutic interventions targeting skin tumors and other diseases using LSOM.In summary, we successfully demonstrate the capability of LSOM for non-invasive longitudinal monitoring of skin vascularization in transgenic VEGF-A mice e.g., 10 × 10 mm² lateral FOV), while zoom-in images close to the ear edge  were recorded with a finer 5 µm step size. The scan parameters of LSOM images and their corresponding scan time are listed in Table Microvascular networks of mouse skin were evaluated by the label-free LSOM imaging technique Figure A, based i.e., hemoglobin in perfused skin blood vessels) with a per-pulse energy < 900 nJ and a repetition rate up to 12 kHz. The generated broadband US signals of vessels were recorded by a spherically focused polyvinylidene fluoride (PVDF) US sensor , which was coaxially and confocally aligned with the GRIN lens. The signals were then amplified by a 24 dB low-noise amplifier  before being digitized by a 2-channel, 250 MS/s, 16-bit data acquisition (DAQ) card . Afterwards, the recorded signals were reconstructed and analyzed using our developed algorithms in Matlab. Due to the low numerical aperture (NA = 0.05) of the GRIN lens, a 7.5 µm spot size with an extended depth of focus > 1 mm was achieved, thus averting the need for optical sectioning and sequential acquisition of images from different depths A 532 nm nanosecond laser  was used to generate laser pulses, while the energy was tuned by a pair of half-wave plate and polarizing beam splitter. Directly attached to a single-mode optical fiber, the custom-designed gradient index (GRIN) lens  was used to focus the light onto the target  were calculated for further statistical analysis of the vascular network  k Figure B.Hemoglobin (Hb) content was calculated based on the sum of raw LSOM image intensity values divided by the total imaged area, aiming to rule out any potential bias introduced by the CLAHE and filtering procedures. Fill fraction - the total blood vessel area divided by the total imaged area - is the most straightforward metric to evaluate the skin vasculature distribution. Number of identified vessels, vessel diameter and vessel tortuosity  were also included into the assessment. The median values of the vessel parameters, either per location or per mouse, were extracted with the mean values ± standard deviations calculated and presented for the LSOM image statistics. The p-value was calculated using an unpaired two-tailed t-test, where p-value < 0.05 was considered as statistically significant and p-value < 0.001 as statistically highly significant. The total time for processing one MIP image was less than 40 s.The following parameters were analyzed for a quantitative comparison between WT and VEGF-A mice: VEGF-A under control of the keratin 14 promoter (FVB1 genetic background) in basal keratinocytes of the skin and other stratified epithelia and in the outer root sheath of the hair follicles, were evaluated for their alterations in the skin vasculature in comparison to their WT littermates ad libitum. Mouse maintenance and all animal experiments of this study were approved by the local veterinary authority .K14-VEGF-A mice, which express  in vivo skin imaging, mice were anesthetized using a mixture of isoflurane and oxygen/air (20/80%) for all experiments. The animals were placed on a self-regulated heating pad at 37 °C  and incubated overnight with goat biotinylated anti-Lyve1  and rat anti-CD31  primary antibodies, followed by donkey anti-rat AlexaFluor 488 and anti-biotin streptavidin AlexaFluor 594 secondary antibodies , and counterstained with Hoechst 33342 (Thermo Fisher Scientific).For immunofluorescence staining of frozen skin samples, 7 µm cryosections were fixed in cold acetone (5 min) and cold methanol , incubated overnight with rabbit anti-Lyve1  and rat anti-CD31  primary antibodies, followed by anti-rat AlexaFluor 488 and anti-rabbit AlexaFluor 594 secondary antibodies (Thermo Fisher Scientific), and counterstained with Hoechst 33342.For ear whole-mount immunofluorescence staining, the ears were split and the cartilage was gently removed. Tissue was fixed with 4% PFA for 2 h at 4 °C and washed with PBS/0.3%Triton X-100. Subsequently, ear skin sections were blocked for 2 h in washing solution containing 0.3% bovine serum albumin and 5% donkey serum and incubated overnight with rabbit anti-Lyve1  and rat anti-CD31  primary antibodies, followed by 2 h washing and 4 h incubation with anti-rabbit AlexaFluor 488 and anti-rat AlexaFluor 594 secondary antibodies . Nuclei were counterstained with Hoechst 33342.+ vessels were controlled for lymphatic vessels by immunostaining for the lymphatic endothelial cell marker Lyve1+. In non-wounded dorsal skin sections, the CD31-positive area was quantified in 1 cm wide representative areas and corrected by the Lyve1-positive area for lymphatic vessels. Ear sections were analyzed at the base, base-middle, middle-tip and tip in a 750 µm wide representative region on the dorsal side. Vessel-like structures were filled, and area fraction was measured per pre-defined ROI and the number of vessels counted.Light microscopy and fluorescence images were acquired with a Pannoramic 250 Slide Scanner  at 20× magnification. Whole-mount ear images were acquired with a Leica TCS SP8 inverted system at 10× magnification. All histological sections were analyzed in Fiji JR, LB, SW, MD and DR conceived the study. JR and YL conducted the longitudinal LSOM experiments. LB and MG conducted ear whole-mounts and classical skin histology imaging. JR implemented the vessel analysis algorithm. LB and YL performed vessel segmentation and analysis. LB and YL prepared the figures. YL prepared the tables. SW, MD and DR supervised the study. All authors contributed to writing and revising the manuscript.


The objective of this study was to compare airway management technique, performance, and peri-intubation complications during the novel coronavirus pandemic (COVID-19) using a single-center cohort of patients requiring emergent intubation.We retrospectively collected data on non-operating room (OR) intubations from February 1–April 23, 2020. All patients undergoing emergency intubation outside the OR were eligible for inclusion. Data were entered using an airway procedure note integrated within the electronic health record. Variables included level of training and specialty of the laryngoscopist, the patient’s indication for intubation, methods of intubation, induction and paralytic agents, grade of view, use of video laryngoscopy, number of attempts, and adverse events. We performed a descriptive analysis comparing intubations with an available positive COVID-19 test result with cases that had either a negative or unavailable test result.We obtained 406 independent procedure notes filed between February 1–April 23, 2020, and of these, 123 cases had a positive COVID-19 test result. Residents performed fewer tracheal intubations in COVID-19 cases when compared to nurse anesthetists (26.0% vs 37.4%). Video laryngoscopy was used significantly more in COVID-19 cases (91.1% vs 56.8%). No difference in first-pass success was observed between COVID-19 positive cases and controls . An increased rate of oxygen desaturation was observed in COVID-19 cases (20.3% vs. 9.9%) while there was no difference in the rate of other recorded complications and first-pass success.An average twofold increase in the rate of tracheal intubation was observed after March 24, 2020, corresponding with an influx of COVID-19 positive cases. We observed adherence to society guidelines regarding performance of tracheal intubation by an expert laryngoscopist and the use of video laryngoscopy. A critical complication of coronavirus 2019 (COVID-19) is acute respiratory insufficiency requiring supplemental oxygen and mechanical ventilation. A recent report from Wuhan, China, found that 14% of patients with novel coronavirus infections developed acute hypoxemic respiratory failure and 2.3% of patients required endotracheal intubation (ETI).Endotracheal intubation (ETI) in suspected COVID-19 cases presents challenges related to rapid patient decline, infection control, and resource scarcity. Several groups in China and Italy have described precipitous decline in oxygen saturation after loss of spontaneous breathing, particularly in patients with decreased respiratory reserve.10Recent expert consensus guidelines from the Safe Airway Society recommend that the airway operator be the most experienced clinician available and, in anticipated difficult cases, the intervention should be performed by an anesthesiologist.First-pass success is an important measure of patient prognosis following ETI.11This is an unmatched retrospective cohort analysis of all ETIs prospectively recorded in a continuous quality improvement database from February 1–April 23, 2020. All patients who underwent out-of-operating room (OR) ETI were included, as the note is specifically used for emergent procedures. This project was granted exemption by our institutional review board as it is an analysis of a quality improvement database. All patient name and health record numbers were made anonymous to the researchers.What do we already know about this issue?Coronavirus disease (COVID-19) prompted changes to intubation processes, primarily aimed at infection prevention. First-pass success rate is an accepted intubation performance indicator.What was the research question?We sought to compare first-pass success intubation rates prior to the COVID-19 pandemic surge to those of COVID-positive cases during the surge.What was the major finding of the study?There was a significant increase in intubations in response to COVID-19 with no difference in first-pass success rate.How does this improve population health?COVID patient intubations can be performed effectively as measured by first-pass success, even with additional infection control and process changes.We conducted this study in a 514-bed academic medical center and safety-net hospital in Boston, Massachusetts. The majority of intubations are typically performed by residents in both the emergency medicine (EM) and anesthesiology residency programs using either a direct or video laryngoscope. All intubations performed in the ED and ICU are supervised by attending physicians. The department of anesthesiology transitioned the airway response team to be comprised of expert laryngoscopists including nurse anesthetists (CRNA), anesthesiologists, and select senior residents. Prior to February 1, 2020, CRNAs did not regularly perform emergent intubations. No changes were made to the constitution of the airway response team in the ED.Multidisciplinary meetings were held on best practice in airway management of COVID-19 patients at our institution in late February 2020. A consensus was reached to include routine use of rocuronium with propofol for longer paralysis to minimize disconnection of the breathing circuit and video laryngoscopy to maximize FPS in suspected COVID-19 patients . All patAn airway procedure note user interface and reporting system was designed with structured and free-text fields integrated within our hospital’s electronic health record, Epic . Specific variables are auto-populated including patient identifier, date-time, author, and specialty. Provider type is collected as anesthesiologist, attending EP, CRNA, or resident. The primary airway operator then must document the following: technique; pre-oxygenation; induction and paralytic agent; C-spine immobilization; laryngoscopist; laryngoscope size; grade of view obtained; external laryngeal manipulation; and common post-procedure complications  .The adverse events in the procedure note coincide with those identified in the NEAR study and were defined according to the intubating clinician’s discretion.We reconciled missing data within the complication reporting section by assigning “no complication” in the absence of documentation to ensure even distribution of under-reporting bias. For this analysis, controls were designated as those with a negative test result and those who did not have a test sent to ascertain disease-specific variation in FPS and complication rates. Given limited testing capabilities early in the pandemic, patients were not universally tested and we assume this group was most similar to test-negative subjects due to low clinical suspicion.We analyzed variables using JMP Pro, version 15 . Data were analyzed by intubation date and both persons under investigation and COVID-19 status. We used descriptive statistics with tests of association to analyze variables according to COVID-19 status. Variables within procedure notes were not mutually exclusive . We therefore chose analyze each specific variable across COVID-19 positive versus control status to compare incidences to avoid introducing bias. Tabulated data is therefore presented as column percentages and raw response rates. Categorical variables were summarized as number (%) and compared using a Pearson’s chi-square test or two-tailed Fisher’s exact test at a two-sided significance level of 0.05. The assumptions for the chi-square test were that the study groups were independent and that the sample size had cell count with >5 cases. Continuous variables are presented as medians and with a 95% confidence interval (CI).A post-hoc sensitivity analysis of minimum effect size (MES) was conducted for the Fisher’s exact test assuming two independent samples  with an α = 0.05 and 1-β = 0.8. For the primary endpoint, FPS rate, we used a previous baseline FPS rate of 84% from unpublished institutional data and 89% from a recent report of emergency tracheal intubation in COVID-19 patients by Yao, Wang, and Jiang et al.We identified 405 discrete procedure notes filed and 350 COVID test results between February 1–April 23, 2020 . On MarcThe majority of intubation procedures in both groups were performed in designated ICU-level areas including OR and post-anesthesia care unit locations rather than the ED. . A face An increased rate of oxygen desaturation was observed  in COVID-19 cases, but there were no significant differences in other peri-intubation adverse events . HoweverWe performed subgroup analysis to characterize trends in intubation performance (FPS), use of video laryngoscopy, and incidence of desaturation by provider types. First-pass success was obtained in 194/224 cases (86.7%) performed by residents compared to 168/182 (92.3%) performed by non-resident providers  . Video lFor a period of 30 days from March 24–April 24, 2020, we observed a doubling in the number of emergent non-OR intubations performed daily at our institution with a majority attributed to COVID-19. We performed a retrospective analysis of all airway procedure notes documented at our hospital and have presented trends in clinical parameters that suggest adherence with recently published guidelines .The role of trainees during the pandemic was the subject of much discussion in our institution as it was internationally. Striking an ethical balance between potential exposure and the need for supervised experiential learning continues to be debated. We observed a decreased proportion of COVID-19 intubations performed by trainees and an increase in those performed by non-trainee providers, particularly CRNAs. This can be attributed to the reconfiguration of the anesthesia emergency airway response team prior to March 24. The ED intubation team remained as an attending and senior airway resident, with attending discretion on laryngoscopist. Of note, both EM and anesthesia attending success rates were the lowest in their fields. This is alarming at first glance, but we hypothesize this was largely due to the attending preferentially performing the more difficult intubations, which included cases in which the patients were profoundly hypoxemic and/or morbidly obese.A higher percentage of intubations involving COVID-19 patients were performed by anesthesia when compared to EM. Since EM, with rare exception, performs all intubations in the ED, this statistic suggests that a higher percentage of COVID-19 patients deteriorated once admitted, and subsequently required intubation. This is congruent with our internal unpublished data that indicates 53% of our COVID-19 ICU admissions originated as transfers from the wards (47% occurring directly from the ED). Although some intubations performed by anesthesia represent repeat intubations such as a tube changes or re-intubation after unplanned/failed extubation, it is unknown whether the incidence of repeat intubations could have been higher in the COVID-19 group. These repeat intubations were not excluded in this study and may present a different likelihood of FPS.We found increased utilization of video laryngoscopy in COVID-19 cases that is consistent with recent expert recommendations.The use of several preoxygenation modalities including high-flow nasal cannula, non-invasive positive pressure ventilation (NIPPV) and bag valve mask (BVM) were linked to aerosolization and increased risk of nosocomial infection during the 2003 SARS epidemic.We attempted to compare unmatched cases by selecting a temporal cohort during which a standardized intubation protocol had been implemented. However, we acknowledge the limitation of retrospective analysis as our methodology.We acknowledge that this study did not involve chart review and that no additional quality analysis was performed to ensure complete capture of emergent intubations or complications during the study period. The authors served as data abstractors in our study and were not blinded to the study hypothesis. A potential under-reporting bias was introduced when missing complication data was assigned to the null. However, we anticipate that this would be evenly distributed among both COVID-positive and negative cases. Additionally, the authors were not blinded during data appraisal. Our study was secondarily limited in that the definition of intubation attempt was not determined a priori, and variation among providers is likely present. For our post-intubation complication rates, reporting bias is possible with complication documentation, as under-reporting in health records is a known phenomenon. The assumption that this bias is equally distributed between COVID-positive and controls was not determined.th, 2020. We found that first-pass success and complications other than oxygen desaturation were not significantly different between COVID-19 positive cases and controls. Future prospective trials should investigate factors surrounding emergency airway management including team composition and video laryngoscopy on intubation performance.We observed a significant increase in the total volume of emergent intubations performed at a single center in Boston, Massachusetts, during the 30-day period March 24–April 24


Gastrointestinal (GI) symptoms are one of the common somatic symptoms presented in patients with major depressive disorder (MDD). Higher frequency of GI symptoms and higher GI symptom burden were linked to greater depression severity and increased risk of suicide ideation. However, few studies have explored the underlying mechanisms of GI symptoms in MDD. Based on previous studies, the cerebellar-DMN circuits may play a potentially critical role in GI symptoms comorbid with depression. Fifty-two first-episode drug-naive patients with MDD (35 with GI symptoms and 17 without GI symptoms) and 28 matched healthy controls were recruited in the current study and underwent resting-state functional magnetic resonance imaging scan. Cerebellar seed-based functional connectivity maps were established. Relative to depressed patients without GI symptoms, significantly increased cerebellar-anterior default mode network (DMN) connectivities were found in those with GI symptoms. Both increased and decreased functional connectivities were found between cerebellum and posterior DMN in patients with GI symptoms compared with those without GI symptoms and healthy controls. Moreover, the right Crus I - right superior temporal gyrus connectivity value was related to severity of GI symptoms and depression in all patients with MDD. The support vector machine analysis demonstrated a satisfactory classification accuracy (89%) of the disrupted cerebellar-DMN connectivities for correctly identifying MDD patients with GI symptoms. These results revealed the possible neural mechanisms for the involvement of cerebellar-DMN circuits in GI symptoms co-occurred with MDD. Major depressive disorder (MDD) is one of the most common and debilitating psychiatric disorders worldwide characterized by heterogeneous symptoms and highly variable course trajectories . BesidesGastrointestinal (GI) symptoms, such as nausea, vomiting and gastric distention, are one of the common somatic symptoms presented in patients with MDD. Approximately 30–70% patients with MDD had concomitant GI symptoms . ConsistIt is well-known that affective disorders are common comorbidities of functional gastrointestinal disorders (FGID). Co-occurrence of depression was found in about 30% patients with FGID in primary care settings and even higher in tertiary care . InteresExtending beyond the motor aspect, cerebellar contributions to cognition and affect have attracted numerous interests over the past decades. Neuroimaging studies revealed a complex configuration of cognitive/affective topographic organization in the cerebellum . GeneralIn the current study, we aimed to explore the intrinsic cerebellar functional connectivity differences across MDD patients with and without GI symptoms and healthy controls using seed-based resting-state functional magnetic resonance imaging (fMRI), and to investigate the possible associations between altered cerebellar functional connectivity and the severity of GI symptoms. Based on previous evidence reviewed above, we hypothesized that there would be disrupted cerebellar-DMN connectivity involved with GI symptoms in patients with MDD. Furthermore, we examined whether the altered cerebellar-DMN connectivities could serve as potential biomarkers for GI symptoms in MDD and reliably discriminate MDD patients with GI features from those without GI symptoms or healthy controls employing support vector machine (SVM) approach.A total of fifty-two first-episode drug-naive patients diagnosed with MDD according to the DSM-5 criteria by professional psychiatrists were recruited from the Second Xiangya Hospital of Central South University, including 35 patients with GI symptoms and 17 patients without GI symptoms. None of the patients had other Axis I or Axis II psychotic disorders. While 28 demographically similar healthy controls without a history or a family history of psychiatric disorders were recruited from the local community. All individuals were 18–55 years old and right-handed. Subjects would be ruled out if they: (1) had acute physical illness  or neurological illness, or a history of substance abuse; (2) had a history of brain injury resulting in loss of consciousness; (3) were pregnant or were unable to undergo MRI scans. All the participants provided a written informed consent, and the study was obtained approval from the ethics committee of the Second Xiangya Hospital of Central South University.Symptom severity was assessed using 17-item Hamilton Rating Scale for Depression (HRSD-17) by a trained clinical psychiatrist. Specifically, items 10, 11, 12, 15, and 17 represent anxiety/somatization factor, item 16 represents weight loss factor, items 2, 3, and 9 represent cognitive disturbance factor, items 1, 7, 8, and 14 represent retardation symptoms, items 4, 5, and 6 represent sleep disturbance, and item 12 represents GI symptoms.2, and acquisition time = 500 s (250 volumes).Magnetic resonance imaging scanning was conducted on a Siemens 3T scanner . During the scanning, all participants were instructed to keep relax, motionless, and keep eyes closed but not fall asleep. Foam pads and earplugs were used to minimize head motion and reduce scanner noise, respectively. Resting-state fMRI data were acquired using a gradient echo-planar imaging sequence with the following parameters: TR/TE = 2,000/30 ms, flip angle = 90°, slice thickness/gap = 4/0.4 mm, number of slice = 30, 64 × 64 matrix, field of view = 240 × 240 mm1 in MATLAB n MATLAB . The foln MATLAB , and sign MATLAB .r-to-z transformation to improve the normality of their distribution. Voxel-wise one-way analysis of covariance (ANCOVA) and then post hoc t-tests were employed to identify inter-group differences in cerebellar functional connectivity maps with age, sex, years of education, and framewise displacement as covariates. Multiple comparisons were corrected using the Gaussian Random Field (GRF) theory with a significant threshold of p < 0.05 .Three cerebellar seeds involved in the DMN were selected as ROIs: left Crus I , right Crus I , and lobule IX . A 6 mm radius sphere was defined as a ROI for each center point using the REST software . We creap < 0.05. All the statistical analyses were conducted by using SPSS version 23.Age, years of education, framewise displacement, and clinical characteristics were compared across three groups by using one-way analysis of variance (ANOVA). Sex differences among groups were tested using a chi-square test. The associations of disrupted cerebellar connectivity values with clinical variables in patients with MDD were examined using Pearson correlation analysis. The significant level was set at We selected the significant inter-group differences in the cerebellar-DMN connectivity as features to discriminate patients with MDD from healthy controls and to classify MDD patients with and without GI symptoms using a Gaussian kernel SVM method in the Matlab. Generally, a well-defined sample was used in SVM to create an optimal hyperplane  that was capable of distinguishing between categories and predicting a new target subject that belongs to which predefined group . We emplAs shown in Compared with healthy controls, MDD patients with GI symptoms had increased functional connectivity between the left Crus I and bilateral precunues and right inferior parietal lobule , betweenr = −0.378, p = 0.006) and HRSD scores , the right Crus I-right precunues , lobule IX-left angular , lobule IX-right angular , lobule IX-right Crus I , lobule IX-left superior frontal gyrus , and lobule IX-right superior frontal gyrus .We extracted the mean functional connectivity values of the significant inter-group differences and examined whether any of them were associated with clinical variables. In all patients with MDD, functional connectivity between the right Crus I and the right superior temporal gyrus was negatively correlated with GI symptoms scores , with a sensitivity of 86% and a specificity of 93%. Similarly, the between-group differences of cerebellar-DMN circuits in G1–G0 comparison could correctly classify depressed patients with GI symptoms into G1 group with an accuracy of 88% (46 of 52 in the two groups), a sensitivity of 94%, and a specificity of 76% .This study focused on GI symptoms in first-episode, drug-naive patients with MDD and preliminarily explored the resting-state cerebellar functional connectivity patterns of these patients. First, consistent with previous studies, more severe depressive symptoms were found in patients with GI symptoms in the psychiatric care settings compared with those without GI symptoms and healthy controls. Our results demonstrated that patients with GI symptoms have more severe anxiety/somatization symptoms, greater weight loss, and more severe sleep disturbances than patients without GI symptoms and healthy controls. Second, significantly increased cerebellar-anterior DMN connectivities were found in patients with MDD accompanied with GI symptoms compared with those without GI symptoms. Both increased and decreased functional connectivities were found between cerebellum and posterior DMN in patients with GI symptoms compared with those without GI symptoms and healthy controls. Moreover, our results revealed that the right Crus I - right superior temporal gyrus connectivity value was related to severity of GI symptoms and depression in all patients with MDD. Third, our SVM analysis further demonstrated a satisfactory classification accuracy (89%) of these disrupted cerebellar connectivities for correctly identifying patients with GI symptoms, which indicated the potential role of cerebellar-DMN circuits in the mechanisms of MDD with co-occurring GI symptoms.As expected, we found that depressed patients with GI symptoms experienced more severe depression and anxiety/somatization. Similar results have also been reported by studies focusing on patients with FGIDs. Compared with patients without FGIDs, prevalence of depression or anxiety was increased in patients with FGIDs, and among them, asymptomatic patients had the lowest prevalence. In addition, the increase of the prevalence of depression and anxiety exhibited in a stepwise manner with the increase of the frequency and/or severity of GI symptoms . The cloThe DMN is a critical large-scale network and encompasses two subsystems (the anterior and posterior part) contributing differently to the pathopsychological characteristics of MDD . The mPFAberrant serotonin (5-HT) function have been found in diverse cerebral regions (including mPFC/ACC) in MDD , and theThe lateral temporal gyrus, parts of DMN, is one of the well-acknowledged areas involved in the pathophysiology of MDD. They play crucial roles in social cognition and emotion regulation . A greatThere are still several limitations in this study. First, the small sample size may affect the reliability of our results. Second, the severity of GI symptoms was evaluated by only one item from the HRSD. Although some previous studies  also empIn conclusion, abnormal cerebellar-DMN functional connectivity was observed in patients with MDD accompanied with GI symptoms. The high classification results between depressed patients with GI symptoms and those without GI symptoms and healthy controls obtained by SVM analysis revealed the possible neural mechanisms for the involvement of cerebellar-DMN circuits in GI symptoms co-occurred with MDD.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Medical Research Ethics Committee of the Second Xiangya Hospital of Central South University, China. The patients/participants provided their written informed consent to participate in this study.All authors contributed to the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Negative symptoms occur in individuals at ultrahigh risk (UHR) for psychosis. Although there is evidence that observer ratings of negative symptoms are associated with level of functioning, the predictive value of subjective experience in daily life for individuals at UHR has not been studied yet.This study therefore aims to investigate the predictive value of momentary manifestations of negative symptoms for clinical outcomes in individuals at UHR.Experience sampling methodology was used to measure momentary manifestations of negative symptoms  in the daily lives of 79 individuals at UHR. Clinical outcomes  were assessed at baseline and at 1- and 2-year follow-ups.b=−4.62, P=.01). Higher levels of anhedonia were associated with poorer functioning at the 1-year follow-up . Higher levels of social anhedonia were associated with poorer functioning  and greater illness severity  at the 1-year follow-up. In exploratory analyses, there was evidence that individuals with greater variability of positive affect (used as a measure of blunted affective experience) experienced a shorter time to remission from UHR status at follow-up .Lack of social drive, operationalized as greater experienced pleasantness of being alone, was associated with poorer functioning at the 2-year follow-up (Targeting negative symptoms in individuals at UHR may help to predict clinical outcomes and may be a promising target for interventions in the early stages of psychosis. Negative symptoms occur in individuals at ultrahigh risk  for psychosis and have been reported to be associated with reduced quality of life and impaired functioning in cross-sectional and longitudinal studies -4. RecenTo date, clinical outcomes in UHR studies have primarily focused on transition to psychosis. Given that most individuals at UHR do not develop psychosis (71%-76%) as indicated in meta-analyses and systematic reviews -16, inveStandard measures used to assess negative symptoms  ,25, thouPrevious experience sampling studies have investigated blunted affective experience, lack of social drive, anhedonia, and social anhedonia as momentary manifestations of negative symptoms in daily life. Blunted affective experience has been operationalized as intensity , instability , and variability  of positive and negative affect ,33-35. LTo our knowledge, only 2 experience sampling studies have, to date, investigated momentary manifestations of negative symptoms in individuals at UHR ,37. AlthThis study aims to investigate whether momentary manifestations of negative symptoms predict clinical outcomes  in individuals at UHR for psychosis at the 1- and 2-year follow-ups. We tested the following hypotheses:Momentary manifestations of negative symptoms in daily life predict clinical outcomes in individuals at UHR at 1- and 2-year follow-up such that higher levels of (1) blunted affective experience ; (2) lack of social drive ; (3) anhedonia ; and (4) social anhedonia  are associated with greater illness severity and poorer functioning at follow-up.In exploratory analyses, we further aimed to examine whether momentary manifestations of negative symptoms are associated with time to transition to psychosis or remission from UHR status.We recruited a sample of individuals at UHR aged 15-35 years, who were assessed at baseline and 1- and 2-year follow-up. Participants were recruited in London (United Kingdom), Melbourne , and Amsterdam and The Hague (the Netherlands) as a part of the high-risk study of the European Network of National Schizophrenia Networks Studying Gene-Environment Interactions (EU-GEI ). EU-GEITo be eligible to participate, individuals had to meet at least one of the UHR criteria as defined by the Comprehensive Assessment of At Risk Mental States : (1) attbeep to complete a brief questionnaire 10 times a day on 6 consecutive days at random moments within set blocks of time.Data on momentary manifestations of negative symptoms were collected using ESM ,41. PartA detailed description of ESM items and compliance procedure is provided in r=0.28, P=.02) and positive affect , variability of negative affect , anhedonia , and social anhedonia . We found no evidence that the BPRS negative symptom subscale was associated with momentary manifestations of negative symptoms. In addition, we used observer-rated measures of negative symptoms to predict momentary manifestations of negative symptoms in a multilevel model (P=.01) and negative affect , instability  and variability  of negative affect, anhedonia , and social anhedonia . The BPRS negative symptoms scale did not predict momentary manifestations of negative symptoms in the multilevel model.Consistent with previous research, psychometric properties for measures of momentary manifestations of negative symptoms were assessed by evaluating their convergent validity. Therefore, we examined the association between momentary manifestations and observer-rated measures of negative symptoms at baseline , the second most significant P value is tested against α=.05/(n−1), the third P value against α=.05/(n−2), and so on. Simes-corrected significant results are highlighted in a footnote in tables.Analyses were adjusted for a priori confounders  or prevalence of comorbid disorders . However, the current sample showed poorer functioning  and lower levels of observer-rated negative symptoms  at baseline. Comparing participants who completed follow-up assessments, the sample with no ESM data collected  showed a lower BPRS total score at 1-year follow-up . There were no significant differences in demographic or clinical characteristics at 2-year follow-up. The ESM sample comprised 79 individuals at UHR, of whom 9 transitioned to psychosis during the study period. Data on clinical outcomes were obtained for 48 individuals at 1-year follow-up and 36 individuals at 2-year follow-up. Participants were on average aged 23 (SD 4.93) years and 56% (44/79) were women. The majority  of the sample was White, followed by 15% (12/79) with Black ethnicity. In addition to their UHR status, 76% (60/79) of the participants were diagnosed with a comorbid axis I disorder . Participants with greater variability were more likely to experience a shorter time to remission from the UHR status. We found no evidence that blunted affective experience predicted transition to psychosis.P=.01 [statistically significant after Simes correction]), such that individuals who experienced greater pleasantness of being alone showed poorer functioning. However, there was no evidence that pleasantness of being alone predicted illness severity and the GAF symptoms score. In exploratory analyses, there was no evidence that lack of social drive predicted time to transition or remission from UHR status.P=.02 [statistically significant after Simes correction]). Lower positive affect in moments of pleasant events or, in other words, higher levels of anhedonia, were associated with poorer functioning. However, we found no evidence that anhedonia predicted functioning at 2-year follow-up. In addition, anhedonia did not predict illness severity at 1- and 2-year follow-up. In exploratory analyses, we found no evidence that anhedonia predicted time to remission or transition to psychosis.P=.045 [statistically significant after Simes correction]) and lower scores on both GAF subscales  at 1-year follow-up. However, we found no evidence that social anhedonia predicted clinical outcomes at 2-year follow-up. In exploratory analyses, we found no evidence that social anhedonia predicted time to remission or transition to psychosis.As displayed in Using an experience sampling design, this study found no evidence that blunted affective experience predicted functioning or illness severity at follow-up (H1). However, there was some evidence that higher experienced pleasantness of being alone was associated with poorer functioning at 2-year follow-up (H2). In addition, our results tentatively suggest that higher levels of anhedonia were associated with poorer functioning at 1-year follow-up (H3). Finally, we found robust evidence that higher levels of social anhedonia were associated with higher levels of illness severity and poorer functioning at 1-year follow-up (H4). In our exploratory analysis, we found no evidence that momentary manifestations of negative symptoms in daily life predicted transition status. However, our results tentatively suggest that blunted affective experience predicted time to remission from UHR status.Our findings should be interpreted in light of several methodological considerations. First, the sample selection should be critically evaluated: ESM is a burdensome research method, which may lead to selection bias, such that individuals with more intense symptoms might be underrepresented in the sample. However, compared with the no ESM sample of the EU-GEI High Risk Study, the participants in this showed comparable levels of illness severity and lower scores on the GAF symptoms subscale at baseline. In addition, the sample showed high comorbidity rates of nonpsychotic disorders, which replicates findings from previous studies and systematic reviews ,54. HighTo our knowledge, this is the first study using an experience sampling design to investigate the predictive value of momentary manifestations of negative symptoms measured in individuals UHR. In accordance with our hypotheses, we found evidence for more intense momentary manifestations of negative symptoms to be associated with poorer functioning and higher illness severity at follow-up. In addition, we found evidence that individuals with greater variability of positive affect (as a measure of blunted affective experience), experienced a shorter time to remission from UHR status. This is in line with findings from previous studies using other operationalizations of negative symptoms -5. GivenOur findings tentatively suggest that blunted affect, lack of social drive, and anhedonia are associated with some clinical outcomes, but findings on social anhedonia were most robust. We may speculate that changes in affective response to social contact  in daily life may be most relevant in individuals at UHR, whereas other types of momentary manifestations of negative symptoms  may be more relevant in later stages of psychosis. Social anhedonia may contribute to a loss of reinforcement of social contact, which might encourage a progressive decrease of social interaction and social functioning more downstream, closer to, or directly at, onset of psychotic disorder ,57-59.The findings have important implications for clinicians and researchers aiming to improve functional outcomes of individuals at UHR. Recent meta-analyses found no evidence for psychosocial treatment to improve functioning in individuals at UHR , with poIn addition, we found only weak correlations between momentary manifestations of negative symptoms and the BPRS scores, highlighting the relevance of participants’ subjective experience. These discrepancies may be interpreted in different ways. First, discrepancies may evolve due to varying modes of assessment and, hence, precision of measurement. Gerritsen, Bagby  claim thWe found evidence for momentary manifestations of negative symptoms, especially social anhedonia, to predict clinical outcomes at follow-up. These findings emphasize that the assessment of momentary manifestations of negative symptoms in individuals at UHR is of considerable potential value for both diagnostic assessment and early intervention. The assessment of momentary manifestations of negative symptoms may provide a more comprehensive picture of patients’ symptoms in the context of their daily life for clinicians and researchers and contribute to a better understanding of individuals’ subjective experience. In addition, the experience of momentary manifestations of negative symptoms, especially social anhedonia, in daily life may be a promising target for interventions aiming to improve clinical outcomes in the early stages of psychosis.


Various neurological manifestations of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported, associated with a broad spectrum of diverse neurological symptoms and syndromes. Estimating rate and relevance of these manifestations remains difficult as there is a lack of standardised case definitions.We defined comprehensive categories including most reported neurological manifestations associated with SARS-CoV-2 to allow for a more standardised data collection. After a literature search of MEDLINE with ten keywords, 12 selected studies and larger case series were included. We compared the rate and relevance of neurological manifestations in hospitalized patients. We propose four main categories including 1) cerebrovascular disease, 2) inflammatory syndromes of the central nervous system (CNS), peripheral nervous system (PNS) and muscle, 3) metabolic/toxic dysfunction of CNS, PNS and muscle and 4) miscellaneous disorders.Ageusia (702) and anosmia (805) have been reported as the most common and the first occurring neurological symptoms. Cerebrovascular disease (451) and encephalopathy (663) were associated with a more severe course and worse clinical outcome. Any neurological manifestation was associated with a longer hospital stay and a higher morbidity and mortality compared to patients without neurological manifestations. We suggest reporting future neurological manifestations of coronavirus disease-19 (COVID-19) following a pathophysiology-based approach using standardized pre-defined case definitions to yield more specific and comparable data. The crisis caused by SARS-CoV-2 has kept the world in suspense since its outbreak in Wuhan, China in 2019. What started as an epidemic was soon declared a pandemic by the World Health Organization (WHO) on the 11th of March 2020 , 36. At In this narrative review we summarise the data on the distribution of neurological manifestations in hospitalized patients, associated with SARS-CoV-2 described in selected literature. We report the rate and relevance of neurological syndromes associated with the virus and suggest a pathophysiology-based approach for the future collection of data on neurological manifestations associated with SARS-CoV-2.SARS-COV-2 is part of the corona virus family comprising seven different virus types that are known to infect humans. It is a large enveloped positive strand-RNA virus with spike proteins on its surface appearing like a solar corona. These surface proteins are used to bind to the human angiotensin-converting-enzyme receptor 2 (ACE2) on human cells and the virus uses the transmembrane protease serine subtype 2 (TMPRSS2) to prime the spike proteins. Therefore, the presence of ACE2 on human cells determines viral cellular tropism. ACE2 is not only expressed in the lung parenchyma, kidney, pancreas, small intestine, testicles and vascular epithelia, but also in the CNS, including neurons and glial cells .Although SARS-CoV-2 primarily presents with respiratory features, several neurological manifestations have been described. This has initiated research on neurotropism of SARS-CoV-2 and resulted in evidence of its capability to infect nerve cells, adding it to a list of neuro-invasive coronaviruses. There are different routes via which SARS-CoV-2 is suspected to infiltrate the CNS including the olfactory route, the trans-synaptic route, the leukocytic route and the haematogenic route  and post-infectious autoimmune effects (cellular immunity and autoantibodies) illustrated in Fig. PNS and muscle can both be affected by direct invasion or systemic immune response. ACE2 is expressed in muscle tissue which presents one possible route of invasion. Peripheral nerves are directly affected and it is postulated that SARS-CoV-2 infection of nervous tissue induces downregulation of ACE2 leading to the overreaction of the Renin-Angiotensin-pathway resulting in neuroinflammation, oxidative stress and vasodilation . SystemiWhile numerous reports and reviews of neurological manifestations of SARS-CoV-2 have emerged to date, the comparability of data across study groups, institutions, and countries remains difficult as different reporting standards are used. Case definitions of neurological manifestations sometimes use a pathophysiology-based approach and at other times use a symptom-based approach, often mixing both approaches. Systematic, standardized case definitions as well as uniform diagnostic criteria of SARS-CoV-2 infections in general and nervous system infection/affection are lacking. Accordingly, overlaps, redundancies, and uncertainties as to which symptom is caused by what type of affection impedes the systemic evaluation of published reports. Without uniform and pre-defined case definitions, the prevalence, incidence, severity, and impact of neurological manifestations of SARS-CoV-2 remain challenging to ascertain. This complicates the development of diagnostic criteria, treatment guidelines and prognostication of neurological affection by SARS-CoV-2 infection.To define neurological manifestations of COVID-19 requires the use of a standardized definition of the clinical association between neurological disease and SARS-CoV-2. The WHO has provided COVID-19 case definitions for confirmed, probable and suspected cases , 36. TheIn the following we propose definitions for conditions included in the main categories.Cerebrovascular disease is defined as a neurologic symptom or syndrome caused by cerebral ischaemia or haemorrhage with a sudden onset of a focal neurological deficit .Encephalopathy describes global brain dysfunction with different underlying pathologies such as metabolic, hypoxic-ischaemic, or septic pathogenesis. No uniform definition exists, and it is not accounted for in formal diagnostic manuals. This issue was addressed by a multi-society consensus paper generating a new recommendation on nomenclature. They define acute encephalopathy as a rapidly (in less than 4 weeks) developing pathobiological brain process expressed clinically as either subsyndromal delirium, delirium, or coma with other possible features such as seizures or extrapyramidal signs. By definition, other terms used to describe encephalopathy such as altered mental state, acute confusional state, acute brain dysfunction and acute brain failure should not be used interchangeably .For delirium we suggest using the five diagnostic criteria as defined in the Diagnostic and Statistical Manual of Mental Disorders. These define delirium as a disturbance in attention that develops over a short period of time with a disturbance in cognition both not better explained by a pre-existing condition. There should also be evidence from the past medical history that the disturbance is a direct physiological consequence of a medical condition .Coma is defined as a state of complete unwakefulness graded using diagnostic systems such as the Glasgow Coma Score .Hypoxic ischaemic brain injury is used to describe diffuse brain injury because of transient anoxia or hypoxia .For CNS and PNS inflammatory syndromes  we suggest using the definitions used by Ellul et a. They define SARS-CoV-2 meningitis, encephalitis, myelitis, or CNS vasculitis as SARS-CoV-2 detected in CSF or brain tissue or evidence of SARS-CoV-2-specific intrathecal antibody and no other explanatory pathogen or cause found. Furthermore, ADEM, GBS and other acute neuropathies associated with SARS-CoV-2 infection are defined as neurological disease onset within 6 weeks of acute infection; and either SARS-CoV-2 RNA detected in any sample or antibody evidence of acute SAR S-CoV-2 infection and no evidence of other commonly associated causes .We defined four categories that included the main neurological complications associated with COVID-19 so far. In the next step we conducted a literature search in order to extrapolate data for our new system of categorisation.n > 30. The literature search was started in December 2020 using the following keywords: neurological, neurologic, nervous system, brain, manifestations, complications, peripheral nervous system, central nervous system, COVID-19, and SARS-CoV-2.In an attempt to comprehensively appreciate reported observations while trying to reduce –not eliminate – redundancies, the following categories were defined; 1) cerebrovascular disease, 2) inflammatory syndromes of CNS, PNS and muscle, 3) metabolic/toxic dysfunction of CNS, PNS and muscle and 4) miscellaneous disorders. Both reported manifestations as well as symptoms and syndromes were assigned a category. If no assignment was possible the manifestation was grouped under miscellaneous disorders. Thereafter, a search was limited to English language manuscripts and to studies and case series with After scanning the abstracts for syndrome categories of neurologic manifestations we developed a table to group the different neurological manifestations using a pathology-based approach. We assessed the rate of neurologic manifestations in relation to the total rate of COVID-19 cases in the respective publication, if reported. Neurologic manifestations such as altered mental state were grouped under encephalitis or encephalopathy depending on the underlying cause. One prospective case series from Singapore described the additional category of autonomic nervous system dysfunction. As this has only been scarcely described in present literature it was not included as a separate category .Cases were excluded if the manifestations were too unspecific such as ‘other peripheral disorders’, ‘headache’, ‘dizziness’ and ‘syncope’. We report the rate of neurological manifestations as percentage per total SARS-CoV-2 infections if recorded and the rate of neurologic manifestations per category as shown in Table The literature search yielded 12 full text articles in English literature describing neurological manifestations and complications of SARS-CoV-2 including a rapid review (901 patients), retrospective as well as prospective observational case series , cohort studies (137 patients) and surveillance studies (153 patients).All case series that were included identified a SARS-CoV-2 infection by a positive reverse transcriptase Polymerase Chain Reaction (rt-PCR) of SARS-CoV-2 from a nasopharyngeal swab. Additionally, some case series chose to utilize the WHO criteria for confirmed, suspected and probable cases. The infection severity of the SARS-CoV-2 associated pneumonia was graded using clinical scores such as the American Thoracic Society score for community-acquired-pneumonia. Stroke severity was sometimes assessed using the National Institutes of Health Stroke Scale and the degree of disability by using the Modified Rankin Scale. The Confusion Assessment Method for Intensive Care Unit was most often used to monitor delirium. Most studies reported clinical neurological, laboratory and radiological findings and, if examined, CSF analysis. Most also reported treatment approach of neurological manifestations associated with SARS-CoV-2 and, if recorded at the time, patient outcome at discharge Fig. .Fig. 3ROur literature search identified 12 studies reporting categorizable neurologic manifestations in patients diagnosed with SARS-CoV-2. The overall rate of neurologic manifestations differed massively between studies. While four studies  did not compare the rate of neurologic manifestations in COVID-19 patients to those without neurologic manifestations, the remaining eight studies reported a rate ranging from 0.08%  to 90% . Nearly COVID-19 patients with neurological manifestations had a higher disease severity and a worse prognosis. In a retrospective observational case study, any neurological symptom was associated with longer hospital stay (8 days versus 5 days). Encephalopathy and stroke were associated with more severe disease, based on the need for mechanical ventilation  . Meppielp < 0.05). Even after adjusting for confounders, COVID-19 patients with neurologic disorders had an increased risk of in hospital mortality  and a decreased likelihood of discharge home  [This could be due to pre-existing morbidity as Frontera et al. describe that patients with neurologic disorders were older, male, white, hypertensive, diabetic, intubated and had higher sequential organ failure assessment scores (< 0.001) . In cont< 0.001) . However< 0.001) .Certain case studies reported a significant association between CVD and abnormal coagulation parameters with raised D-dimers, fibrinogen, and prolonged Partial Thromboplastin Time in combination with thrombocytopenia , 23, 24.3, presence of compatible acute lesion on brain MRI, rt-PCR positive for SARS-CoV-2 in CSF’ [Inflammatory CNS syndromes were rarely reported. For encephalitis, the diagnosis was either based on pathology or clinical presentation. Several pre-existing diagnostic criteria were used such as encephalitis defined by clinical consensus criteria , 28, cri in CSF’ . Ellul e in CSF’ , 36. To  in CSF’ . In the  in CSF’ . Additio in CSF’ .Treatment approaches included corticosteroids, hydroxychloroquine, intravenous immune globulin (IVIG), convalescent plasma, interferon beta and several antiviral drugs  , 21, 23.The most reported manifestation was GBS with 65 cases. The diagnosis of GBS was mainly based on existing diagnostic criteria such as the Hadden criteria or the Brighton diagnostic criteria. The diagnosis was verified by albumino-cytological dissociation as well as typical demyelinating patterns in the electromyography in several studies , 19, 23.Several disorders were reported under the category of skeletal muscle inflammation including rhabdomyolysis (54/378), myalgia (251/378) and elevated Creatine Kinase (CK) levels in the serum (73/378). All studies reported a raised serum CK in association with raised C-reactive-protein (CRP), myoglobulin and lactate dehydrogenase.Encephalopathy was defined differently in each study owing to the general lack of a standardized definition. Most definitions were symptom-based or partly symptom- and pathology-based, encompassing an alteration in the level of consciousness in the absence of signs of CNS inflammation . Only tw. Two studies reported a mortality of 15% [Several studies point towards an association between CVD and more severe diseasey of 15%  and 38.5y of 15%  for SARSy of 15% .Outcomes were variable and often incomplete as most studies were published without follow-up data. One study reported a mortality of 4.8% and a recovery rate of 47% . Ellul eIn the rapid review of Ellul et al. outcomes were split with one third receiving physiotherapy, one third being discharged and one third still ventilated at the time of publication . In a rep = 0.03). However, uncertainty remains as to more than one symptom per patient was counted and therefore the primary manifestation could have been a different one with rhabdomyolysis being the result on the initial presenting complaint or a complication of ICU stay [Treatment was reported as supportive in one study with good outcome as symptoms improved . In contICU stay .Ellul et al. reported 49 patients with encephalopathy requiring an ICU stay and only one patient who did not. Liotta et al. described an overall longer hospital stay in patients with encephalopathy. In the prospective study of Frontera et al., encephalopathy was the most common diagnosis and especially common among patients with severe COVID-19 . As to oEncephalopathy was associated with worse outcome due to multifactorial reasons. Fan et al. report that patients with delirium could not tolerate non-invasive mechanical ventilation and were therefore referred to the ICU for invasive mechanical ventilation . Liotta In this review we categorized neurologic manifestations of COVID-19 as reported in larger case series and noteworthy studies. We found that neurologic manifestations are generally associated with a higher morbidity and mortality as well as a worse outcome. Even though the exact rate of neurologic manifestations in patients with COVID-19 remains unclear it is vital to raise clinical awareness in order to recognize and treat patients early and adequately.One explanation for the high discrepancy between the rate of neurological manifestations in COVID-19 patients ranging from 0.08 to 90% could be the generosity of case descriptions. If cases were pre-defined the rate was naturally much lower than if cases were included based on a non-systematic subjective description derived from medical records. Secondly the rate of neurologic manifestations is strongly dependent on the population that is examined, such as in Helms et al’s cohort where only patients admitted to ICU due to severe ARDS where included in contrast to Koh et al’s cohort where all microbiologically confirmed COVID-19 patients in Singapore where included , 14. We Even though PNS disorders including the presentation with anosmia and ageusia were quantitatively the most frequent manifestations, encephalopathies and cerebrovascular disorders are of more systemic relevance due to a higher association with morbidity and mortality. Encephalopathy in general is a misfortunately chosen term as by definition it means that there is a malfunction of the brain which is neither a pathophysiologic nor a clinical definition and its use certainly remains problematic. Whether anosmia and ageusia are always SARS-CoV-2-related remains questionable and until proven otherwise this generous association should be made with caution. The differentiation between local epithelial, PNS or CNS affection underlying these symptoms remains similarly challenging. Nevertheless, these were reported as the earliest neurologic symptoms of SARS-CoV-2 infections and any patient presenting with these features warrants investigation for the disease.An important issue raised by a review on neurobiology of COVID-19 applies to the proportion of neuro-inflammatory syndromes that are caused by direct viral affection of COVID-19 versus those that are caused by para- or post-infectious effects e.g. coagulopathy or endothelial inflammation . This isThis review has several limitations. Considerable studies did not provide detailed enough data to compare distinct parameters across different studies which decreases the validity of such comparison. For example, the “break-through” publication on the topic by Mao et al. only provide little insight into their approach of categorization and describe several CNS manifestations such as cerebrovascular disease, dizziness, headache, impaired consciousness, ataxia and seizures as one group without differentiating between symptoms, syndromes and diagnoses .Altered mental state was often accounted for as a presenting symptom and studies handled this very differently. Varatharaj et al. divided the presenting symptom of altered mental status between encephalitis, encephalopathy or neuropsychiatric disorders depending on the assumed underlying pathology . MeppielThe retrospective multicentre case series by Meppiel et al. was one of the few studies that actively excluded other possible aetiologies besides SARS-CoV-2 for encephalopathy and clearly defined neurological manifestations a priori . LikewisThis narrative review offers an approach to categorization of neurological manifestation based on several big case series. Robust estimation of the situation is strongly affected by discrepancies in case definitions, mixing symptoms/syndromes with diagnostic findings, differences in patient cohorts, and lack of systematic exclusion of alternative causes of neurologic disease. Further prospective studies are needed to better understand the underlying pathophysiology of neurological manifestations in COVID-19 disease including case control studies to ascertain the actual rate and relevance of such manifestations. These studies need systematic definitions of manifestations such as those proposed by us or used in very few existing studies , 19. FurIn conclusion, all reviewed studies demonstrate that neurological manifestations are broad and heterogenous suggesting different underlying pathogenic processes and pathways. It is crucial to explore the causality of SARS-CoV-2 regarding neurological manifestations and to exclude likely alternative causes. Therefore, clearly defined, standardized, and universal case definitions should be used across countries. Even though neurologic manifestations might represent a relatively small part of COVID-19 phenomena, early recognition and treatment is key to prevent a more severe outcome associated with higher morbidity and mortality.