Is successful percutaneous revascularization of chronic total occlusions associated with improved quality of life?This meta-analysis of 7 trials, including 2500 patients, found that successful chronic total occlusion revascularization was associated with improved quality of life parameters of patients compared with patients receiving optimal medical therapy or after failed chronic total occlusion revascularization.This study’s results may help physicians in their decision-making process when treating this high-risk group of patients. This systemic review and meta-analysis investigates whether there is an association of successful chronic total occlusion percutaneous revascularization with quality of life by analyzing the relevant domains of the Seattle Angina Questionnaire. Chronic total occlusion percutaneous coronary intervention (CTO-PCI) is not usually offered because of skepticism about long-term clinical benefits.To assess the association of successful CTO-PCI with quality of life by analyzing the relevant domains of the Seattle Angina Questionnaire (SAQ).PubMed, EMBASE, Web of Science, Google Scholar, and Cochrane databases were searched to identify randomized trials and observational studies specifically addressing quality of life domains of SAQ from January 2010 to June 2022.Studies included reporting SAQ metrics such as angina frequency, physical limitation, and quality of life, before and after CTO-PCI.The present study was performed according to the Cochrane Collaboration and Preferred Reporting Items for Systematic Reviews and Meta-Analyses statements, in which fixed-effect or random-effect models with generic inverse-variance weighting depending on statistical homogeneity were applied. Data were extracted by 3 independent reviewers.The primary outcome was angina frequency; physical limitation and quality of life were assessed as secondary outcomes.P = .002; I2 = 86.4%) and they experienced less physical activity limitation , and greater quality-of-life domain  compared with patients with optimal medical therapy or failed procedure. Furthermore, follow-up duration  was associated with a significant decrease in angina frequency in meta-regression analysis.Seven prospective randomized or observational studies (2500 patients) were included, with a mean (SD) participant age of 61.2 (2.1) years. CTO-PCI was associated with significantly improved quality-of-life metrics during a mean (SD) follow-up of 14.8 (16.3) months. In patients with successful procedures, angina episodes became less frequent (mean [SD] difference for SAQ angina frequency of 12.9 [3.1] survey points ; standardized mean difference was 0.54 ; In this systematic review and meta-analysis examining quality of life following CTO-PCI, successful procedures were associated with improved quality-of-life parameters compared with patients on optimal medical therapy or after failed CTO-PCI. These findings suggest support for using PCI to treat CTOs in symptomatic patients unresponsive to medical treatment. Landmark studies in patients with CTO have suggested no difference in mortality between patients treated with PCI compared with OMT, although randomized trials searching for hard outcomes of CTO-PCI are strongly hampered by selection bias and low enrollment.10 Recently, American College of Cardiology/American Heart Association/Society for Cardiovascular Angiography and Interventions 2021 Coronary Revascularization Guideline’s class of recommendation on CTO treatment has been downgraded from IIA to IIB based on 2 randomized trials showing no improvement in left ventricle function following revascularization with a neutral effect on hard outcomes.11Chronic total occlusion (CTO) is reported in 15% to 25% of patients with stable coronary artery disease (CAD).12 in a substudy of the ISCHEMIA trial,13 otherwise perceived as a negative trial in terms of hard clinical outcomes for revascularization.14 In addition, quality of life scales are generally accepted as critical patient-reported outcome metrics for evaluating interventions in stable CAD.16 Demonstrating comparable symptom relief and quality of life improvement with percutaneous CTO treatment can support the reappraisal of the class of recommendation and underscore the potential of CTO-PCI revascularization to improve patients’ health status.There is general agreement that the primary benefit of revascularization in chronic coronary syndromes is symptomatic improvement, convincingly confirmed using the Seattle Angina Questionnaire (SAQ)17 Therefore, we conducted a systematic review and meta-analysis including all available trials assessing the life quality using validated SAQ metrics to test whether successful CTO-PCI is associated with improved quality of life.A recent review article studied patients’ quality of life (QoL) following CTO revascularization and reported a trend of improvement in QoL indices.PRISMA) reporting guideline, and the Meta-analysis of Observational Studies in Epidemiology (MOOSE) checklist used for observational studies.20 The study protocol was registered in the PROSPERO database before starting (CRD42022349139).This meta-analysis study was performed according to the Cochrane Collaboration, Preferred Reporting Items for Systematic Reviews and Meta-analyses , with differences resolved by consensus. We included published studies that compared the QoL outcomes using SAQ metrics with successful vs failed PCI or OMT alone among patients with CTO. If more than 1 study reported outcomes of the same cohort, we included the most recent or most comprehensive study. We excluded all papers studying non-CTO lesions or treatment with coronary artery bypass grafting (CABG). Patient and lesion characteristics were extracted on a per-protocol analysis, including age, gender, cardiovascular risk factors, SAQ metrics score, and medication. The observational studies identified with this selection process were also assessed for eligibility based on full-text review following the MOOSE checklist22 The scores of each dimension were analyzed separately. Although variations existed between the follow-up duration, SAQ metrics were reassessed at baseline and follow-up.The primary outcome was angina frequency (AF) as defined in the SAQ-AF. SAQ physical limitation (SAQ-PL) and SAQ-QoL were assessed as secondary outcomes. Ordinal scores on the questionnaire were transformed to a continuous scale, from a low score of 0 to a maximum score of 100.The quality of included studies was independently appraised by 2 authors . In addition, for each included study, we evaluated the risk of bias  for random-sequence generation, allocation concealment, blinding of patients and physicians, blinding during the follow-up assessment, preliminary outcome evaluation, and selective reporting, in keeping up with the Cochrane Collaboration approach.P < .10 and based on the Cochran Q test, with heterogeneity score (I2) values of 25%, 50%, and 75% representing mild, moderate, and severe heterogeneity, respectively.Continuous variables were reported as mean (SD) or median (IQR). Categorical variables were expressed as number and percentage. Statistical pooling for incidence estimates was performed using a fixed-effect or random-effect model with generic inverse-variance weighting depending on statistical homogeneity, computing risk estimates with 95% CIs using Comprehensive Meta-Analysis software version 3.0 (Biostat Inc). A comparison between successful CTO-PCI, OMT, and failed CTO-PCI groups was performed regarding SAQ-AF, SAQ-PL, and SAQ-QoL in both randomized and observational trials separately. These outcomes were analyzed as continuous variables and the difference between baseline and last follow-up was assessed as standardized mean differences (SMDs). Results were reported as SMDs between groups with 95% CI. In addition, meta-regression analysis was performed to determine the importance of baseline features on the primary outcome and leave-one-out analysis to evaluate any single study effect. Hypothesis testing for statistical homogeneity was set at 2-tailed 27 were eligible for inclusion in this meta-analysis, 3 were prospective randomized trials and 4 were prospective observational studies. We used the NOS to evaluate the 7 included studies as described previously. Detailed scores are presented in the eAppendix in The flowchart for the search strategy and study inclusion is presented in P = .002; I2 = 86.4%) between successful CTO-PCI and combination of failed PCI and OMT. Additionally, meta-regression analysis was performed using the baseline SAQ-AF, which was not found significant (P = .01) was associated with a significant improvement in SAQ-AF. Age, gender, and medications such as calcium canal blockers, β-blockers, and nitrates, did not have an association with results.Angina frequency was significantly improved following successful CTO-PCI in 5 out of the 7 contributing studies (2500 participants). The mean (SD) difference for SAQ-AF was 12.9 (3.1) survey points  and SMD was 0.54    difference of 9.7 (6.2) survey points  and SMD of 0.42  B. The Qo= 58.8%) C.I2 = 49% for successful PCI-CTO and OMT comparison, and I2 = 91% for the successful and failed PCI comparison. The pooled main outcome of PCI-CTO and SAQ-AF in RCTs was a risk ratio (RR) of 0.40  and in observational data it was an RR of 0.51 . The pooled main outcome of PCI-CTO and SAQ-PL was an RR of 0.27  in RCTs, and in observational data it was an RR of 0.43 . The pooled main outcome of PCI-CTO and SAQ QoL in RCTs was an RR of 0.43 , and in observational data it was an RR of 0.41 . There were no interactions between study designs.Four studies  compared successful CTO-PCI with OMT. The other 3 studies compared successful vs failed CTO-PCI. The subgroup analysis for each treatment was performed to further show the association of successful CTO-PCI with the SAQ . As in tThe bias evaluation confirmed a high-quality level for all studies included in the primary analysis  are critical components to assessing whether health care services and practices make a difference to a patient’s life. PROMs are an assessment of health or well-being from the patient’s perspective without interpretation by a clinician or health practitioner collected through patient-reported outcome measures.32 Small trials managed to use objective measurable changes in exercise performance. The IMPACTOR-CTO trial  found a significant reduction in ischemic burden with an improvement in 6-minute walk distance.9 In a study conducted by Sheehy et al,33 78% of the patients were angina-free at 6 months following CTO-PCI. Our study adds to the literature by using the well-validated SAQ questionnaire. To our knowledge, this is the first meta-analysis to formally establish the association of successful CTO-PCI with functional and health status.A growing body of evidence indicates benefits, such as symptom improvement and reduced long-term health care costs following CTO-PCI.37 A different outcome cannot be excluded if a true all-comers trial with sufficiently long-term follow-up is conducted sticking to a strict policy not allowing crossover. In practice, such a trial is unlikely to be feasible and the ethics of a trial forcing half of patients randomized to OMT to live with disabling symptoms for years are questionable. We probably have to accept that, similar to other PCI in other chronic coronary syndromes, the only proven benefit of CTO-PCI is symptom relief. Treatments based on health status–related life quality are therefore becoming more critical in medical decision-making and are recommended by guidelines to aid in treatment strategies for CAD.15Most of the randomized CTO-PCI studies focused on hard end points, and this approach was encouraged by the results of some registries showing a survival benefit.The higher procedural and in-hospital complications rate could have determined the worse long-term outcome of patients with failed procedures. This led us to perform a substudy dividing the comparison groups into patients remaining in OMT with no attempt to CTO-PCI and patients with failed CTO-PCI. No differences were observed regarding symptomatic improvement during follow-up, suggesting that the worse outcome was not driven by compromise of collateral flow or other periprocedural complications at the time of the initial CTO-PCI.10 As a long-term follow-up study, patients with successful CTO-PCI had an improved physical activity, lesser angina frequency, and greater treatment satisfaction compared with patients with failed CTO-PCI at a 4-year median follow-up.23 A large CTO-PCI randomized trial (DECISION-CTO) studied the association of CTO-PCI with life quality8 using the SAQ and showed a significant improvement at least at 30 days. Similarly, EURO-CTO trial evaluated life quality metrics among patients whose symptoms were deemed to be genuinely attributed to CTO and showed a consistent significant superiority of CTO-PCI over OMT alone.27 Furthermore, there are meta-analyses that have shown successful CTO-PCI is associated with less residual angina in the literature.39 The most recent 1 by Abuzeid et al,17 revealed symptomatic improvement following CTO-PCI via either PCI or CABG, and showed improvement in the quality of life. Our current analysis found results in the same direction. However, there are crucial differences: (1) in order to avoid using the same patient group, subgroup analyses were not included, (2) other crucial SAQ metrics were analyzed besides the QoL parameter, (3) only studies that compared successful or unsuccessful CTO-PCI or OMT groups were picked up, and (4) patient groups with CABG treatment were excluded. In this way, we aimed for less margin of error by analyzing more homogeneous patients.It was clearly observed that the quality of life and physical activities were associated with a gradual improvement in patients successfully revascularized with CTO, even in the studies that demonstrated worse or neutral MACE rates.10 In patients with concurrent CTO in the left anterior descending artery, left ventricle ejection fraction was significantly higher in the CTO-PCI group. Based on cardiovascular physiology, it is not perceived as a surprising finding to see such an improvement following revascularization.41 It is a phenomenon called collateral (or coronary) steal that appears when the vasodilatory reserve is limited and defined as an absolute or relative fall in coronary blood flow to a vascular region in favor of another supply area under conditions of hyperemia,42 which is undoubtedly the case of CTO vessel. The recent study analyzed the flow alternation in the collateral circulation in patients with CTO, before and after successful revascularization.40 A significant reduction was shown in collateral flow over time following successful CTO-PCI.It is important to realize that there is a complex interaction between the CTO vessel and the other coronary arteries through the collateral channels that are present. Therefore, successful results were associated with improved myocardium globally. The EXPLORE trial randomized survivors of a STEMI within 7 days to get PCI or conservative management of a concomitant CTO to observe the alteration in left ventricle ejection fraction at 4 months.There are some limitations to this study. First, each study carries inherent selection bias invariably associated with patients’ characteristics, CTO site, and time to last SAQ assessment. Second, before-after SMDs have some intrinsic limitations because the baseline and posttest scores are not independent. However, the between-group SMDs  partially overcame this limitation because they reduced the association of these unmeasurable variables with results at least for the baseline evaluation. Another limitation was that we showed absolute improvement in patients’ life quality using SAQ, instead of using certain thresholds for SAQ domains. The reason was the lack of patient-based data so we could not find patients with AF less than 60. Showing absolute improvement will be correlated much better with long-term outcomes, because some patients with successful CTO-PCI feel better due to physical improvement over time. Therefore, trapping between certain thresholds would limit our long-term estimate. Additionally, the sensitivity analyses and the meta-regression results might have been affected by the small number of the included studies and unmeasured confounding factors and must be interpreted with caution.This systematic review and meta-analysis of patients included in 7 randomized trials and registries found a clear improvement in health status–related life quality as assessed by the validated SAQ metrics in patients receiving a successful CTO-PCI. These findings suggest support for using PCI to treat CTOs in symptomatic patients unresponsive to medical treatment.


To advance understanding of early childhood bed-sharing and its clinical significance, we examined reactive bed-sharing rates, sociodemographic correlates, persistence, and concurrent and longitudinal associations with sleep disturbances and psychopathology.Data from a representative cohort of 917 children (mean age 3.8 years) recruited from primary pediatric clinics in a Southeastern city for a preschool anxiety study were used. Sociodemographics and diagnostic classifications for sleep disturbances and psychopathology were obtained using the Preschool Age Psychiatric Assessment (PAPA), a structured diagnostic interview administered to caregivers. A subsample of 187 children was re-assessed approximately 24.7 months after the initial PAPA interview.Reactive bed-sharing was reported by 38.4% of parents, 22.9% nightly and 15.5% weekly, and declined with age. At follow-up, 48.9% of nightly bed-sharers and 88.7% of weekly bed-sharers were no longer bed-sharing. Sociodemographics associated with nightly bed-sharing were Black and (combined) American Indian, Alaska Native and Asian race and ethnicity, low income and parent education less than high school. Concurrently, bed-sharing nightly was associated with separation anxiety and sleep terrors; bed-sharing weekly was associated with sleep terrors and difficulty staying asleep. No longitudinal associations were found between reactive bed-sharing and sleep disturbances or psychopathology after controlling for sociodemographics, baseline status of the outcome and time between interviews.Reactive bed-sharing is relatively common among preschoolers, varies significantly by sociodemographic factors, declines during the preschool years and is more persistent among nightly than weekly bed-sharers. Reactive bed-sharing may be an indicator of sleep disturbances and/or anxiety but there is no evidence that bed-sharing is an antecedent or consequence of sleep disturbances or psychopathology.The online version contains supplementary material available at 10.1186/s13034-023-00607-w. Bed-sharing, defined as a child and parent sleeping in the same bed for part or all of some or all nights, is a practice that has varied widely around the world and throughout human history . In the Although prevalence and developmental trends in US bed-sharing during early childhood are not well-characterized, several themes emerge across available studies. First, bed-sharing in early childhood is relatively common. In the 1980 and 1990 s, several studies found 41–55% of children between the ages of 6 and 48 months shared a bed with a parent at least occasionally . Second,Parents and pediatric healthcare professionals may question whether bed-sharing is associated with developmental risk or harm after infancy. Multiple studies have documented concurrent associations between bed-sharing and sleep problems, particularly night waking and bedtime resistance , 13–15. intentional bed-sharing that is chosen proactively by parents due to parenting preferences and/or cultural norms and reactive bed-sharing that is a response to factors such as child behavior or limited resources [Combining key bed-sharing subtypes may contribute to these mixed findings. Conceptual models of bedsharing –23 distiesources , 24. In esources  and may esources , night-tMethodological limitations also contribute to the mixed findings. First, most studies have assessed bed-sharing using a single questionnaire or interview item , 8, 26, To advance understanding of early childhood bed-sharing (after infancy) and whether it is a cause for concern in the US, we focused on reactive bed-sharing and examined (1) prevalence and associated sociodemographic factors, (2) developmental trends, and (3) concurrent and longitudinal associations between bed-sharing and sleep disturbances and mental health diagnoses. We explored sociodemographics correlates  of reactive bed-sharing to provide preliminary information about potential cultural variation in bed-sharing and to inform directions for future research on culturally-sensitive family sleep guidance. In our longitudinal analyses, we addressed prior methodological weaknesses by controlling for baseline symptomatology and exploring directionality.The Duke Preschool Anxiety Study is a screen-stratified study of 917 preschool children recruited from Central North Carolina primary care pediatric clinics. The original aims of the study were to characterize the prevalence, comorbidities and risk factors of preschool anxiety disorders. Therefore, children who screened high for anxiety were over-sampled, a procedure used to ensure that an adequately representative sample of children with anxiety disorders was obtained to address the primary goals of the project. To establish prevalence rates from a multistage sampling design, subjects were assigned a weight inversely proportional to their probability of selection. The original study was approved by Duke University School of Medicine Institutional Review Board (IRB).Children ages 2 through 5 years attending the Duke Children’s Pediatric Primary Care Clinics were screened for study eligibility from January of 2007 to October of 2010. Of the 3433 children screened, 943 (27.5%) screened high and 2490 (72.5%) did not screen high for anxiety using the Child Behavior Checklist (CBCL) for Ages 1½–5 anxious/depressed scale. A cutpoint based on data from an earlier study  was usedOf the 1132 children selected to participate, 1113 were eligible. Inclusion criteria for screening were (1) the child was 24–71-months-old, (2) attended the clinic during the screening period, and (3) a parent/legal guardian was present and consented to screening. Exclusion criteria were (1) lack of a parent/legal guardian with adequate fluency in English, (2) the child was known to have intellectual disability (IQ < 70), autism, or other pervasive developmental disorders, (3) the child’s sibling was already participating, (4) the child was not accompanied by a legal guardian who could provide consent, or (5) the child was considered by the provider to be too medically ill on the day of screening to participate. Nineteen (1.7%) met exclusion criteria and 196 (17.6%) parents/legal guardians chose not to participate. Informed consent forms were signed by a parent or legal guardian by 917 (82.4%) of eligible parents prior to each phase of the study including screening, and interviews. PAPA assessments took place over 47 months (January 2007–December 2010).A follow-up assessment was completed with 187 of the original participants as part of a neuroimaging study. Children with anxiety disorders were oversampled (representing two-thirds of the cohort), and the remainder of the sample were healthy controls from the original cohort. Informed consents were signed and PAPA interviews were completed again approximately 24.7 months after initial assessment. More detail on study design is available in previous publications –31.Preschool Age Psychiatric Assessment (PAPA). The PAPA, a parent-report interview that assesses psychopathology in 2- to 5-year-olds, is based on the parent version of the Child and Adolescent Psychiatric Assessment [sessment . The PAPsessment .Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition but also other diagnostic classification systems applicable to young children such as the Research Diagnostic Criteria: Infancy and Preschool [The PAPA evaluates diagnoses drawn primarily from the reschool . Once threschool . Diagnosreschool .A bed-sharing variable was created using a series of questions in the sleep section of the PAPA. Parents were first asked whether their child slept with a family member for part of the night or the whole night due to reluctance to sleep alone. If this behavior was present, frequency, duration and onset were obtained. Frequency of bed-sharing was used to create three categories: (1) None/Rare: no bed-sharing or rare bed-sharing, 2) Weekly: bed-sharing at least once per week but less than every night, and 3) Nightly: bed-sharing every night. Parental reported, interview based assessments of bed-sharing and classification by bed-sharing frequency are consistent with prior literature , 7, 11. Sociodemographic information, obtained from the background section of the PAPA,was also examined including child age, sex and race and ethnicity, parental education level, and poverty. Race and ethnicity was categorized as follows: Black/African American, Hispanic, White, and (combined) Asian, American Indian, Alaska native, Native Hawaiian, Other Pacific Islander, and 2 or more races. Caregivers could mark off all categories applicable to the child. The assessment of poverty was determined by referencing the family’s reported income level against the annual US federal guidelines for poverty thresholds given the number of individuals in the family .All analyses were conducted using weighted logistic and poisson regression in the SAS Version 9 Software procedure GENMOD. Odds ratios are provided for dichotomous outcome variables and means ratios for Poisson distributed scales. To account for sampling procedures, participants from all samples were assigned a weight inversely proportional to their probability of selection to account for screen-stratification. Thus, results from all analyses reported here are intended to represent unbiased estimates for the original primary care population from which the sample was drawn. Sandwich type variance corrections were applied to adjust for the parameter and variance effects induced by the sampling stratification.Bivariate analyses involved prediction of sociodemographic and psychopathology variables by dummy-coded independent variables representing each frequency of bed-sharing . For analyses predicting psychopathology status, models were adjusted for significant covariates including age, parental education, race and ethnicity, family poverty status and other diagnoses. In parallel analyses, we substituted dummy variables for psychopathology with counts of symptoms in each category. Longitudinal analyses were adjusted for a similar set of covariates as well as time since initial interview and status of outcome variable at baseline.Bed-sharing rates and associations with sociodemographic factors are presented in Table After controlling for sociodemographics and other forms of psychopathology, multivariate ordinal logistic regression analyses indicated that only anxiety disorder status was associated with nightly bed-sharing and only sleep disturbance status was associated with weekly bed-sharing. Parallel analyses with continuous symptom variables revealed the same pattern. These results are depicted in Table Table p = 0.26).Among those who reported nightly bed-sharing at baseline, only 41.3% continued to report nightly bed-sharing at follow-up, while 9.7% reported less frequent bed-sharing (at least weekly) and 48.9% reported no bed-sharing. Thus, nightly bed-sharing declined but did not disappear. In contrast, weekly bed-sharing at baseline declined markedly to the point that 88.7% reported no bed-sharing at all at follow-up. These results are very similar to rates with no bed-sharing at baseline, as 89.9% of these parents continued to report no bed-sharing at follow-up. Overall, the odds of nightly bed-sharing at follow-up were 32 times higher  if nightly bed-sharing was reported at baseline compared to no bed-sharing. Bed-sharing was not associated with presence of follow-up visit  was a common practice in early childhood with over 1 in 3 parents reporting some bed-sharing and over 1 in 5 reporting nightly bed-sharing. However, prevalence varied significantly by sociodemographic factors, with higher rates in families characterized with lower income and educational attainment, and in families reporting child race and ethnicity of Black and (combined) American Indian, Alaska Native and Asian. Bed-sharing prevalence also decreased over development with lower rates among older compared to younger preschoolers. Cross-sectional associations suggested higher levels of separation anxiety and sleep terrors in children who bed-share nightly, and difficulty maintaining sleep and sleep terrors in children who bed-share weekly. Similar associations were observed in longitudinal models. However, associations attenuated after controlling for sociodemographics, time between interviews, and baseline status of the outcome. Our findings suggest reactive bed-sharing is a common practice in families with young children that varies by sociodemographic factors, declines over time, and is a more meaningful indicator of current problems than a risk factor for later struggles.Our overall reactive bed-sharing prevalence was slightly lower than the 41–55% reported in some previous studies , but quiSociodemographic factors were associated with bed-sharing. Consistent with previous research , 10, 11,We were also interested in the clinical significance of preschool-age bed-sharing. At baseline adjusting for co-morbidities, weekly bed-sharing was associated with sleep terrors and difficulty staying asleep, and nightly bed-sharing was associated with separation anxiety and sleep terrors. Our findings are consistent with prior work indicating a fairly robust concurrent link between bed-sharing and sleep problems , 13, 14 12 or 5 11 years of age [Cross-sectional examination of bed-sharing does not address developmental trends. In our sample, bed-sharing decreased but did not disappear from the initial assessment when children were on average 3.8 years old to the follow-up assessment approximately 2 years later. At follow-up, only 41.3% of parents who initially reported nightly bed-sharing and only 11.3% of parents who initially reported weekly bed-sharing continued to do so. These findings are commensurate with previous research indicating that bed-sharing increases after infancy , is modes of age , 42, 43.s of age .Tests of longitudinal associations found no evidence that reactive bed-sharing preceded or followed sleep disturbances and psychopathology. Thus, although bed-sharing in early childhood may be an indicator of sleep disturbances such as sleep terrors and difficulty staying asleep  and/or aThis study had several strengths including a diverse, well-characterized sample, longitudinal design, and use of a structured diagnostic interview. There were also several limitations. First, all information about bed-sharing and psychopathology came from parent report and was derived from a set of items from the PAPA, an instrument which has good psychometric properties overall  and goodIn many respects, our findings suggest that bed-sharing in early childhood (2–6) is predominantly a normative, transient sleep practice influenced by sociodemographic context. Thus, when parents have questions or concerns about bed-sharing after infancy, pediatric providers can discuss with parents the evidence that bed-sharing is relatively common experience in this developmental period and one that may be influenced by family circumstances and cultural context. They can also note factors that may contribute to this increase in bed-sharing such as an increase in nighttime fears and heightened distress about being alone at night. Finally and perhaps most importantly, clinicians can reassure parents about the lack of association between preschool bed-sharing and clinically significant sleep or mental health problems as well as the likelihood that such bed-sharing will decrease, on its own, with time.Supplementary material 1: Table S1. Adjusted associations between Bed-Sharing Status and Sociodemographic Factors


Functional dissociation of brain neural activity induced by opening or closing the eyes has been well established. However, how the temporal dynamics of the underlying neuronal modulations differ between these eye conditions during movement-related behaviours is less known. Using a robotic-assisted motor imagery brain-computer interface (MI BCI), we measured neural activity over the motor regions with electroencephalography (EEG) in a stroke survivor during his longitudinal rehabilitation training. We investigated lateralized oscillatory sensorimotor rhythm modulations while the patient imagined moving his hemiplegic hand with closed and open eyes to control an external robotic splint. In order to precisely identify the main profiles of neural activation affected by MI with eyes-open (MIEO) and eyes-closed (MIEC), a data-driven approach based on parallel factor analysis (PARAFAC) tensor decomposition was employed. Using the proposed framework, a set of narrow-band, subject-specific sensorimotor rhythms was identified; each of them had its own spatial and time signature. When MIEC trials were compared with MIEO trials, three key narrow-band rhythms whose peak frequencies centred at ∼8.0 Hz, ∼11.5 Hz, and ∼15.5 Hz, were identified with differently modulated oscillatory dynamics during movement preparation, initiation, and completion time frames. Furthermore, we observed that lower and higher sensorimotor oscillations represent different functional mechanisms within the MI paradigm, reinforcing the hypothesis that rhythmic activity in the human sensorimotor system is dissociated. Leveraging PARAFAC, this study achieves remarkable precision in estimating latent sensorimotor neural substrates, aiding the investigation of the specific functional mechanisms involved in the MI process. Brain-computer interfaces (BCIs) have emerged as a reliable assistive and rehabilitative technology with proven clinical efficacy for cognitive and motor skills enhancement in healthy subjects ,2 and paOn the other hand, there is a growing body of literature that suggests brain neural oscillations are strongly modulated by the subject’s eye conditions ,16. AlphAlthough extensive research has been carried out on resting-state brain activation in EC and EO, scant attention has been paid to the influence of the eyes’ conditions on the modulations of the sensorimotor cortex during the performance of a movement-related task ,29. HoweTherefore, using a single-case longitudinal paradigm, this study attempts to shed light on neural processes underlying MI with EO and EC during BCI-based post-stroke rehabilitation using EEG. Due to the neuroplasticity characteristic that enables the reorganization of neural pathways in the brain , it is htensor refers to an N-way or  array characterized by more than two modes. If multidimensional data follow a low-rank tensor approximation, they can be represented by higher-order tensors and compressed into multiple unique components with distinct modalities using tensor decomposition methods [N-way tensor decomposition approaches, and its promising applicability in modelling EEG data has been undertaken through a wide range of clinical [In signal processing, the term  methods . Paralle methods  is one oclinical ,42, cognclinical ,44, and clinical ,46,47 stAccordingly, this study leverages PARAFAC to precisely analyze underlying neural mechanisms reflecting MI with eyes-open (MIEO) versus eyes-closed (MIEC), thereby providing deeper insights into differential oscillatory dynamics associated with MIEO and MIEC. Ultimately, these findings have the potential to refine and optimize rehabilitation protocols, presenting new possibilities for the design of personalized, effective approaches, tailored to post-stroke patients.It should be stressed that the raw data used in this study come from the existing dataset collected at the Institute of Measurement Science of the Slovak Academy of Sciences (IM SAS). The EEG data of a male subject (58-year-old), whose left frontotemporal to parietal brain regions were damaged due to an ischemic stroke, have been analyzed in this study. At the beginning of the rehabilitation course, clinical assessments using the Fugl-Meyer Assessment (FMA) , ModifieRelax command). The second voice cue (Move command) was then indicated when the patient had to begin the MI task  and ended (post-training) with two minutes of resting-state EEG recordings with closed eyes, followed by two minutes with open eyes. Between the two pre- and post-training blocks, the subject performed 20 trials of a movement imagination task using a robotic-assisted MI BCI, during which the conditions of MIEC and MIEO were alternating for every 10 trials. Each trial started with a 21-second relaxation period  and right (FC4/C2/C4/C6/CP4) motor cortices as well as the left occipital lobe (O1), referenced to A1 using g.GAMMAcap . The ground electrode was placed at Fpz. The electrode arrangement was in accordance with the 10-5 EEG electrode placement system . All impFor pre-processing, EEG data were firstly down-sampled to 128 Hz. In the first step, a threshold-based automated algorithm implemented in BrainVision Analyzer 2 software (Brain Products GmbH) was employed. The artefact-inclusion criteria involved setting a maximum allowed voltage step of 50 μV/ms, a minimum allowed activity of 0.5 μV in intervals of 100 ms, and a maximum allowed voltage difference of 50 µV in intervals of 20 ms. If any of these criteria were met, the interval preceding and following the detected artefact by 150 ms was marked as bad, except for the third criterion where the interval was set to 50 ms. After the automatic artefact detection, the EEG data were visually inspected using the same software by an expert who manually marked additional periods with undetected artefacts. The artefact markers that were incorrectly assigned automatically, including the detection and removal of ocular artefacts, were also removed by the expert. More details regarding our pre-processing step have been presented in our previous studies . After pN-way tensor N matrices:PARAFAC decomposes an N is the number of modes in the tensor decomposition. Each matrix F represents the number of factors or latent components. The vector ith column of jth mode and N-way super-diagonal configuration with non-zero elements only on the main diagonal. The tensor In the given tensor equations, the symbol A(n) see . This foMatlab by the APECSgui toolbox [N-way toolbox [With the aim of detecting dominant motor-related EEG sensorimotor oscillations for each training day and for each eye condition (MIEC or MIEO), the collected EEG data were modelled separately by a 3-way (time × electrode × frequency) PARAFAC see . To this toolbox , includi toolbox .tripleC method [According to C method , the numC method  followedC method ,47.atoms and how strongly each EEG atom is present over time is termed time scores [For generating time scores, the dominant PARAFAC spatial and spectral weights of all available training sessions for each MIEC and MIEO condition were first averaged together to obtain general atoms. The general spatial and spectral atoms were then projected to the oscillatory EEG spectrum estimated for two-second long overlapping segments with a sliding step size of 7.8125 ms . In other words, the projection was performed for each EEG sample, resulting in the generation of a smooth numerical sequence called time score, in which each value of the time score represents the presence of a specific atom (in the EEG spectrum) and space at a given time. For each MIEO or MIEC condition, a distinct time score was obtained. It is worth clarifying that in this study, narrow-band EEG rhythms are termed e scores ,46,47.Matlab (R2019b) [Statistical analysis was performed using (R2019b) . We cons(R2019b) . The cluThe raw spectrum, non-oscillatory, and oscillatory components were computed by IRASA for each channel, BCI training session, and MI condition. To this end, the MI data were segmented into four-second sliding windows with an overlap of 500 ms, resulting in a frequency resolution of 0.25 Hz. The number of analyzed segments per session per condition after data cleaning was: 860.4 ± 50.9 and 892.7 ± 52.0 (mean ± SD) for the MIEC and MIEO conditions, respectively.Using the three-way PARAFAC, we identified seven dominant general frequency atoms with a clear unimodal structure, which were consistent and relatively repeatable across training days. These atoms closely corresponded to the underlying lateralized spatial signatures presenting cortical sensorimotor EEG oscillations over the left and right hemispheres. As shown in unable to hit the robotic splint). We believed that even in the unsuccessful trials, the patient was engaged in the MI task, but his efforts were not enough to get the defined threshold and control the robotic splint. Meng et al. has also demonstrated this case [The first approach to investigating the modulatory difference of sensorimotor rhythms underlying MIEO and MIEC is to compare the evoked oscillatory activity in movement preparation and movement initiation time frames. In this first step, for calculating time score averages, we considered all MI trials recorded throughout all rehabilitation sessions, regardless of whether they were successful or unsuccessful  and contralateral (left) sensorimotor cortices to smooth the data and gain a maximal signal-to-noise ratio. Here, we empirically selected the z-score method among various methods that exist for baseline correction . FirstlyFor this purpose, the ipsi- and contralateral time scores of each identified frequency atom representing narrow-band EEG oscillatory rhythms were segmented into 14-second epochs. In other words, each epoch included a time frame of 2 seconds before the movement preparation onset .In the movement preparation phase, there was a strong enhancement in 15.5 Hz atom over both ipsi- and contralateral sensorimotor cortices for MIEC compared to MIEO. In contrast, the baseline-corrected time score of 9.5 Hz atom ipsilateral to the hand used for imagery showed a significant decrease for MIEC compared with the MIEO during the movement preparation phase. Applying cluster-based permutation statistics (using time as the clustering dimension) showed significant clusters between 4550 and 1500 ms , where the statistically significant clusters for decreases in oscillatory rhythm time scores were identified in the 8.0 Hz  hand, either with closed- or open eyes. For each eye condition, we assessed the number of successful MI attempts and the duration of each successful MI process as the behavioural performance index. A Chi-Square test was performed to determine whether the proportion of MI duration (the time between imagery onset and triggering the robotic splint) in each histogram bin that reflected a reaction time index, was equal between both different eye conditions. In successful trials, the minimum and maximum MI times were 2.5 and 19.5 seconds, respectively. Whereas, in unsuccessful trials in which the patient failed to hit the robotic splint, the MI time was 20 seconds. The percentage of unsuccessful trials was approximately 52% and 66% for the MIEC and MIEO conditions. There was a significant difference between these two conditions (We used EEG recordings of a stroke patient during longitudinal MI BCI training to assess how cortical sensorimotor oscillations might be modulated by manipulating eye conditions while he imagined moving the affected hand. To this end, we sought to propose a comprehensive mathematical framework based on multiway tensor decomposition to extensively study narrow-band sensorimotor EEG rhythm interactions during MI with EO and EC. In this approach, the multi-channel EEG signal was modelled separately by the three-way PARAFAC to obtain unique time-space-spectral signatures for each training day and eye condition. Individual spatial and spectral characteristics of the identified signatures were then averaged to create a general representation of the most dominant cortical motor-related sources that are active during the MI task. By projecting obtained general weights to the log-transformed oscillatory part of the EEG spectrum recorded, we reliably identified a set of time scores related to these spatial and frequency signatures. Finally, non-parametric, cluster-based permutation statistics were applied to the average of those time scores in order to reveal differential dynamics in sensorimotor modulations with EC and EO during different MI time frames, including movement preparation, movement initiation, and movement completion.We found that the oscillatory neural activity during MIEC and MIEO was modulated differently, providing additional evidence that closed-eyes and open-eyes are fundamentally different behaviours ,29. WhenFirst of all, it should be stressed that, despite the similarities or dissimilarities between the results presented in the current study and those outlined below, we cannot directly compare the result of the current study with the previous research. The previously published results are based on relatively wide-band frequency ranges in the raw EEG spectrum, which may combine multiple oscillatory peaks at a particular spatial position . These aThe suppression of mu rhythm has traditionally been thought to be primarily an index of human mirror neuron system activity when subjects engage in a motor-related task ,66,67. HThis has largely been confirmed in our study, where similar EC and EO oscillatory 8.0 Hz dynamics were observed during the different phases of MI. By contrast, opening the eyes blocked 9.5 Hz desynchronization and changed its dynamic patterns, while 9.5 Hz modulations in the EC condition still represented significant desynchronization relative to the baseline (The stronger contralateral 8.0 Hz desynchronization underlying MIEO  suggestsEvidence from previous neuroimaging recordings in humans  and monkRecent studies have also identified a distinct role for sensorimotor rhythmic activities in the human motor cortex, in which the ipsilateral alpha-band oscillatory power was increased during the stimulation of a movement, whereas the contralateral beta-band oscillatory power was concurrently decreased ,81. HereK factors not being fully inherent in a PARAFAC model with In tensor analysis, identifying the optimum number of factors as a crucial parameter that might severely impact the decomposition quality, is still under debate. A recent study comparing factor number selection methods for PARAFAC decomposition in simulated EEG signals  revealede DBScan  algorithHere, we focused primarily on the oscillatory motor-related EEG rhythms. Neural oscillations co-existing with ubiquitously present non-oscillatory activity are thought to be generated by distinct neural mechanisms ,86. TherBehavioural analysis showed that task performance was affected by eye conditions during the MI process. In our study, the subject performed significantly more successful MI efforts with less MI time during the course of rehabilitation while his eyes were closed. This result supports a recent study showing that while performing a voluntary isometric flexion task, a significant behavioural improvement was observed when subjects had to keep their eyes closed . MoreoveThis study provides empirical evidence that neural oscillations in the sensorimotor cortices are differentially modulated by the simulation of movements with different eye conditions. The use of PARAFAC enables the simplification of the sophisticated longitudinal EEG-BCI data structure by reducing its dimensionality and providing a compact, informative, and physiologically interpretable framework. Despite the highly promising findings of this study, it is necessary to replicate the study on a larger pool of subjects containing both stroke patients and healthy subjects. However, our findings pave the way for the use of multidimensional signal processing frameworks to enable further research into latent mechanisms underlying movement neurophysiology and refine the current outlook on the human sensorimotor system.


Absence status epilepticus (ASE) is the most common type of status epilepticus in patients with idiopathic generalized epilepsy (IGE). Like absence seizure, ASE is characterized by generalized spike-and-wave discharges (GSWDs) on the electroencephalogram (EEG). Once considered specific for IGE, GSWDs have increasingly been observed in other forms of epilepsy, as well as in patients with no prior epilepsy. Here, we report three patients with different types of nonconvulsive status epilepticus (NCSE) in which the EEG correlate was GSWDs: a 44-year-old woman with juvenile absence epilepsy who manifested ASE, a 73-year-old woman with anoxic brain injury complicated by NCSE with well-formed GSWDs (as seen in IGE and de novo ASE), and a 41-year-old woman with frontal lobe epilepsy who developed focal NCSE with impaired consciousness. Evidently, patients with generalized epilepsy, focal epilepsy, and no prior epilepsy can all manifest NCSE with similar electroclinical characteristics, i.e., GSWDs and impaired consciousness. This observation adds to the existing evidence that seizures, whether classified as focal or generalized, often involve focal and generalized elements in their pathophysiology. To fully understand seizure pathophysiology, we must steer away from the focal-versus-generalized paradigm that has dominated the nosology of seizures and epilepsy for a very long time. Generalized spike-and-wave discharges (GSWDs) are seen in the electroencephalogram (EEG) of patients with idiopathic (genetic) generalized epilepsy (IGE) . GSWDs mThe International League Against Epilepsy (ILAE) proposed a quadriaxial system for classifying status epilepticus . Status Here, we present a report on three patients whose EEG showed GSWDs during an episode of NCSE: a 44-year-old woman with juvenile absence epilepsy who manifested ASE, a 73-year-old woman with anoxic brain injury complicated by de novo ASE, and a 41-year-old woman with frontal lobe epilepsy who developed focal NCSE with impaired consciousness.Patient 1 was a 44-year-old woman with intractable juvenile absence epilepsy. At the age of 13, she started having absences and myoclonic seizures and, at the age of 19, she also started having generalized tonic-clonic seizures. She tried almost all antiseizure drugs in various combinations and at maximal dosages and had been on vagus nerve stimulation (VNS) therapy since the age of 22. Despite these measures, she continued to have absence seizures every day, myoclonic seizures every few weeks, and generalized tonic-clonic seizures two to three times a year. Deep brain stimulation was being considered, so she was admitted to the epilepsy monitoring unit (EMU). While off most of her antiseizure drugs, her EEG showed 2.5-3 Hz GSWDs that gradually became more frequent and longer in duration. On day 2, she lapsed into a state of inattention and intermittent unresponsiveness. She was also constantly blinking. Her EEG showed continuous 2.5-3.5 Hz GSWDs with waxing and waning amplitude for about 52 minutes  score of 6. EEG was immediately recorded, and this initially showed well-formed 2.5-3 Hz GSWDs consistent with de novo ASE , and occasional vocalizations, with or without impairment in awareness. She also had generalized tonic-clonic seizures, but this became less frequent with adjustment of her antiseizure drug regimen. She tried almost all antiseizure drugs in various combinations and at optimal dosages. She has also been on VNS therapy since the age of 29. Despite these measures, she continued to have breakthrough seizures almost every month. During her EMU evaluation, while off her antiseizure drugs, she had a focal frontal lobe seizure that evolved into a secondary generalized tonic-clonic seizure. Her interictal EEG showed focal spikes over the left and the right frontal head regions. The next day, her EEG showed sustained 2.5-3 Hz GSWDs  GSWDs appears to be rare after cerebral anoxia. We are aware of only two published reports of de novo ASE in the setting of an anoxic brain injury ,40. In bThe case of Patient 3 presents yet another challenge in choosing a status epilepticus diagnosis. An axis 1 diagnosis for Patient 3 would be “focal frontal lobe NCSE without coma with impaired consciousness.” Although precise, such a long-winded diagnostic term is not pragmatic because it highlights the focal and ignores the generalized aspects of NCSE. Patient 3 is a case in point; she had focal epilepsy, but the onset of NCSE was unmistakably generalized, with GSWDs appearing abruptly in the EEG. A term that does not emphasize the focal or generalized aspects of NCSE may be more appropriate, for example, “dialeptic status epilepticus” . This neIn this observational study, three different types of NCSE, ASE in a patient with IGE, NCSE in coma (with EEG features of de novo ASE) in a patient with no prior seizure or epilepsy, and NCSE with impaired consciousness in a patient with frontal lobe epilepsy, showed GSWDs in the EEG. This is yet another evidence of the fine line between absence and focal epilepsy. Absence seizures are considered generalized but have been shown many times to originate from a cortical focus. Frontal lobe seizures are focal by definition but may generalize rapidly without any trace of focality. Focal NCSE with impaired consciousness involves activation and/or deactivation of the default mode network in both hemispheres. It is time that we dismantle the stronghold of the focal-generalized dichotomy on the nosology of seizure and epilepsy.


Gallus gallus), enzootic nasal tumour virus in goats (Capra hircus) and several small, circular, Rep-encoding, ssDNA (CRESS DNA) viruses in several mammal species. More significantly, we demonstrate that the mNGS method is able to detect potentially lethal animal viruses, such as elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus) and the newly described human-associated gemykibivirus 2, a human-to-animal cross-species virus, in a Linnaeus two-toed sloth (Choloepus didactylus) and its enclosure, for the first time.Metagenomic next-generation sequencing (mNGS) is receiving increased attention for the detection of new viruses and infections occurring at the human–animal interface. The ability to actively transport and relocate this technology enables in situ virus identification, which could reduce response time and enhance disease management. In a previous study, we developed a straightforward mNGS procedure that greatly enhances the detection of RNA and DNA viruses in human clinical samples. In this study, we improved the mNGS protocol with transportable battery-driven equipment for the portable, non-targeted detection of RNA and DNA viruses in animals from a large zoological facility, to simulate a field setting for point-of-incidence virus detection. From the resulting metagenomic data, we detected 13 vertebrate viruses from four major virus groups: (+)ssRNA, (+)ssRNA-RT, dsDNA and (+)ssDNA, including avian leukosis virus in domestic chickens ( Emerging and re-emerging viral pathogens are being observed at an increasing rate. These pathogens can result in the spread of disease to both humans and other animals, as was the case with the zoonotic COVID-19 pandemic . ActivitMetagenomic next-generation sequencing (mNGS) is a powerful technology for identifying new or unexpected pathogens . An impoVulpes vulpes), rodents and urban avian populations, are naturally drawn to zoos due to food availability and shelter. This has also created transmission routes as seen with, e.g., poxviruses, avian influenza and herpes virus transmission, from urban wildlife to rare zoo-housed exotic animal species that has, in some cases, been fatal and detrimental to species conservation [Zoological facilities most commonly exhibit multiple species of animals from more than one continent or biome. Daily husbandry, veterinary procedures, and interactions with the public or staff inevitably impose direct and/or indirect contact between species of animals that would never occur in their natural habitat. Several zoos have recently reported cases of SARS-CoV-2 in carnivores, artiodactyla and non-human primates that most likely occurred by transmission from humans . In addiervation ,16,17,18We previously developed a metagenomic microarray method for the unbiased detection of all known virus genomes described from animals and man . HoweverSamples were collected from 37 different species in Copenhagen Zoo between September and November 2022 . SamplesAll fresh samples were processed on the day of collection. Swabs and biopsies were submerged within cryotubes containing PBS (700 µL) and shaken for 2 min. Aliquots were stored at −80 °C, or mixed 1:1 with MagNA Pure lysis binding buffer . The guanidine iso-thiocyanate in the MPLB-buffer effectively inactivates viruses and proteins, including enzymes, and preserves RNA and DNA . The remAll samples found positive by mNGS for any vertebrate viruses were also analysed with an in-house panvirus microarray assay for supporting findings ,22. The At the time of collection, samples were mixed, 1:1, with MPLB-buffer and processed for analysis. Briefly, nucleic acids from the samples were extracted and purified on a MagNA Pure 96 instrument  and eluted in a 50 µL elution buffer from the MagNA Pure 96 DNA and Viral NA Small Volume Kit . Samples (20 µL) were used for separate random whole-genome transcriptome amplification (WTA) and whole-genome amplification (WGA) according to the REPLI-g Cell WGA & WTA kit protocol . WTA- and WGA-amplified cDNA and DNA, respectively, were purified with the QiaAmp DNA mini-kit  and 1.5 µg cDNA/DNA was labelled with either Cy3 or Cy5 using the SureTag DNA labelling kit . Labelled samples were hybridized to the panvirus microarray (SurePrint G3 Custom Gene Expression Microarray (4 × 180 K), Agilent Technologies, Santa Clara, CA, USA), and the microarray data were processed as described previously ,22.The samples were pretreated according to the field-deployable method described by Fomsgaard et al.  using anRandom amplification was performed with the REPLI-g Cell WGA & WTA Kit , according to the manufacturer’s instructions but with the following alterations: (1) magnetic glass particles (MGPs) were kept in the input material , (2) theOxford Nanopore technology was used for fast and portable sequencing. Up to seven samples were multiplexed using the Rapid Barcoding Kit  without any bead-purification steps for protocol simplicity and a fast library prep of 10 min. Using a Qubit Fluorometer , total DNA concentration for each sample was normalised to contain a maximum 400 ng of DNA before barcoding, as recommended for library preparation. We used R9.4.1 flowcells on the MK1C device  with default settings and fast base calling enabled. The Minion MK1C was used in combination with two power banks that allowed for 10 h real-time sequencing.Choloepus didactylus) was subsequently sequenced using the Nextera XT DNA Library Prep Kit (No. 15031942) to evaluate the presence of human-associated gemykibivirus 2 that was detected with the mNGS method.A nasal swab from a Linnaeus’s two-toed sloth  with a minimum quality score of 7 at each read’s end, as previously recommended by Geneious Prime for metagenomic-generated Nanopore reads and with read lengths above 100 nt. To be able to characterize thousands of reads in a less time-consuming and computational labour-intensive manner, the trimmed reads were BLASTed against the curated offline vertebrate virus Virosaurus database  [The fastq data files were de-multiplexed using MinKNOW (version 21.02.1) and then quality-trimmed using BBduk (version 38.84) using Geneious Prime  . This tr020_4.2) . For theAlignments were manually inspected, and mappings with evenly distributed horizontal coverage of a reference genome were considered true hits for an identified virus. Mappings only consisting of stacked reads in a small region of a reference sequence were considered false hits. To address the incomplete diversity of viruses in Virosaurus, the contigs generated from each mapping were BLASTed using the NCBI nt/nr database and inspected manually for the match between contigs and the reference sequences in order to find possible viruses not present in the Virosaurus database. If viruses scored higher in Bit-score than the Virosaurus-detected virus, the references of these viruses were collected, and the quality trimmed reads were re-mapped to the new reference, and the coverage, depth and virus speciation were inspected to determine whether they improved.For gemykibivirus-positive samples, a phylogenetic analysis was performed on consensus sequences derived from three samples from the same animal see  with genIn total, 130 samples were collected , 15 fluid samples and 3 tissue samples). Some same species and enclosure swabs were sub-pooled . A totalCholoepus didactylus) was sedated for the treatment of lower limb lacerations inflicted by a conspecific. A pooled oral and nasal swab was taken in which a human-associated gemykibivirus  was detected. In total, 96.6% of the reference genome coverage was obtained  enclosure matched multiple metagenomic-assembled genomes (MAG) or partial coding regions of viruses from the family Picornaviridae , chicken picornavirus 4 isolate 5C (Acc. No. KF979335), Melegrivirus A (Acc. No. KF961188) and megrivirus C , which is thought to be a naturally occurring recombinant picornavirus with parts of its genome derived from Melegrivirus A, but has been classified as a megrivirus [Picornaviridae in both healthy groups and groups with runting–stunting syndrome or enteritis [Viral contigs generated from enclosure swabs collected in the chicken  genome. The microarray system also detected a weak positive signal for other avian leukosis/sarcoma viruses , indicating either a coinfection or that distinction between these closely related viruses was unattainable. ALV has a positive-sense RNA genome (7.6 kb) and is naturally occurring, worldwide, in chickens and other Galliformes [Avian leukosis virus (ALV) was detected in the same enclosure swab samples from the chicken coop as described above, with reads assigned to ALV and the chicken . The genomes are 88.8% identical; so, to investigate mis-assignment, the mNGS reads were remapped to JSRV and ENTV-2. The contigs derived from the read mapping to the JSRV reference genome were once more not assigned to JSRV but to ENTV-2. Individuals infected with ENTV-2 can shed the virus through nasal secretions, which was the material in which the virus was detected in this study. Detecting ENTV-2 in this study was not surprising, since it is naturally occurring in goats worldwide except in Oceania [The Enzootic nasal tumour virus (ENTV-2) of goats and the Jaagsiekte sheep retrovirus (JSRV) were detected with the mNGS method and confirmed with the microarray system in a sub-pool of nasal swabs from three goats. However, no contigs derived from either alignment of reads to ENTV-2 or JSRV reference genomes were assigned to JSRV in the NCBI nr/nt database. Both viruses are members of the  Oceania . JSRV no Oceania ,38, and  Oceania ,37,39.Elephas maximus) that had died from haemorrhagic disease. Both the mNGS protocol and the microarray system detected EEHV-1 in two of these cases (Elephant 2 and 3), whereas EEHV-4 was only detected with the mNGS protocol. This is surprising, considering the high viral load in both the serum sample and pericardial fluid identified by an EEHV-specific qPCR (Elephant endotheliotropic herpesvirus (EEHV) 1 and 4 were detected in frozen samples from three juvenile male Asian elephants  and a replication initiator protein [Circoviridae, Genomoviridae and Smacoviridae. Since this study focused on vertebrate viruses, the presence of microalgae-infecting Bacilladnaviridae and plant-infecting Geminiviridae and Nanoviridae was not investigated.CRESS  DNA viruses are a group of viruses that have been detected in a diverse group of hosts  and in environmental samples. They are characterized by their small ssDNA genome size (range of 1–6 kb), fast substitution rates  was detected from the nasal swabs obtained from asymptomatic goats in the Children’s Zoo with complete genome coverage (−8 substitutions/site/year [coverage . Despiteite/year ). Such aite/year . CyCV Eqite/year . HoweverGenomoviridae. Four viruses of the Genomoviridae family were detected: sewage-associated gemycircularvirus-4, faeces-associated gemycircular virus 17, MAG: Genomoviridae sp. isolate ctba76 and human-associated gemykibivirus 2 (Nanoviridae family [ivirus 2 . Sewage-ivirus 2 . This vie family . SaCM-4 Faeces-associated gemycircular virus 17 (FaGmV-17) was detected in a nasal swab from a single horse. The virus was not detected eight weeks later when the same horse was sampled again. It was first detected in chicken faecal swabs in New Zealand but its host specificity is not known and it has not been linked to any clinical disease . The chiGenomoviridae sp. isolate ctba76 was detected in nasal swabs from three horses and one pig with full-genome coverage (Genomoviridae sp. isolate ctba76 (Acc. No. MK032755). Remapping the reads for each sample improved the reference coverage to 100% for all four samples. The virus was originally identified in environmental swabs in and around wild mice in the USA [Genomoviridae sp. isolate ctba76 is not associated with any known disease and is therefore not a concern currently, but its presence in several different mammals could indicate that this virus might be more ubiquitous than previously considered.The virus MAG: coverage . The vir the USA . As rodeGenomoviridae could not be detected due to the absence of probes for these viruses.The microarray system detected GemyCV-SL1 in the sloth , while tSmacoviridae. Viral contigs derived from a fresh faecal sample from a juvenile male Lar gibbon (Hylobates lar) showed high similarity with Gorilla-associated porprismacovirus 1 with 52.6% horizontal reference coverage. It was first described from a gorilla stool sample in San Francisco Zoo alongside other CRESS DNA viruses in other non-human primates [primates . It is uprimates . Howeverprimates , and anoprimates  arguing The mNGS field method and the microarray system both use the REPLI-g with the phi29 enzyme for random nucleotide amplification as do other virus metagenomics studies ,61. The Rousettus aegyptiacus), which the mNGS method did not detect. Subsequent real-time qPCR assays for human rotavirus A produced negative results for oral swabs  but confirmed the presence of rotavirus A in rectal swabs (CT = 31.7) and serum (CT = 31.9). Although the human rotavirus A qPCR could confirm some of the microarray findings, this qPCR may not be optimal for the quantification of bat rotaviruses. Both the mNGS method and the microarray system were developed for the detection of viruses in individuals with clinical symptoms, with the assumption that the viral load would be higher during disease and thus more readily detected [The microarray system detected 7 of the 13 viruses identified by the mNGS protocol, while 6 viruses were not identified. In four cases, this was potentially due to degraded frozen samples, but also decreased probe specificity or absent probes. However, the microarray system did detect multiple different group A rotaviruses from different host species in three samples  from a sub-pool of four Egyptian fruit bats (detected . The reldetected .The use of MPLB buffer for hand-held NA extraction was initially developed for the targeted detection of Ebola virus RNA in whole blood and urine samples . In our During this two-month study, we did not aim to sample all 230 different animal species in the Copenhagen Zoo . The colFor the further application of this mNGS field protocol, we suggest the use of this protocol in geographical regions where advanced diagnostic laboratory infrastructure is lacking and where biodiversity hotspots with an increased risk of viral pathogen spillovers are present.With an optimized mNGS method for fast virus detection using a portable system, this study set out to investigate which viruses were present or circulating in Copenhagen Zoo as a test-of-concept. Thirteen different vertebrate viruses, representing four Baltimore classification groups, were detected. While the presence of virus commonly occurring in host species, such as AVL in chickens and ENTV-2 in goats, was expected, the presence of viruses, such as CRESS DNA viruses, were interesting and their abundance could indicate that these viruses are part of our normal environment and hence also in a zoological facility. It was, furthermore, shown that the mNGS method was readily able to detect potentially lethal animal viruses, such as EEHV in Asian elephants as well as the newly described GemyCV-SL1, a human-to-animal cross-species virus, in a Linnaeus two-toed sloth for the first time. It is envisaged that this portable mNGS system will allow for rapid on-site virus identification and thus may help to mitigate large outbreaks.


Antibacterial nonwoven fabrics, incorporated with Ag, have been applied as masks and air conditioner filters to prevent the spread of disease from airborne respiratory pathogens. In this work, we present a comparison study of Ag ions: Ag and AgCu nanoparticles (NPs) coated onto nonwoven fabrics intended for use as air conditioner antibacterial filters. We illustrate their color changes and durability running in air conditioners using antibacterial activity testing and X-ray Photoelectron Spectroscopic (XPS) analysis. We found that AgCu NPs showed the best antibacterial efficacy and durability. XPS analysis indicated that the Ag concentration, on both the AgCu and Ag- NP-coated fibers, changed little. On the contrary, the Ag concentration on Ag ion-coated fibers decreased by ~30%, and the coated NPs aggregated over time. The color change in AgCu NP-coated fabric, from yellow to white, is caused by oxide shell formation over the NPs, with nearly 46% oxidized silver. Our results, both from antibacterial evaluation and wind blowing tests, indicate that AgCu NP-coated fibers have higher durability, while Ag ion-coated fibers have little durability in such applications. The enhanced durability of the AgCu NP-coated antibacterial fabrics can be attributed to stronger NP–fiber interactions and greater ion release. Over the last two decades, the incorporation of Ag-based nanoparticles (NPs) and ions has been widely employed in the textile and health care industries ,2,3,4,5.Apart from the monometallic nanoparticles applied as textile finishing agents, bimetallic NPs have received much interest because of their optical, electrical, magnetic, and catalytic capabilities, and especially their excellent antibacterial properties, which differ dramatically from their monometallic counterparts in most circumstances ,26. BimeThe durability of an antibacterial fiber is associated with its application requirements  filters are extremely efficacious at screening most bacteria and viruses due to their tiny pores (which may be up to 100 nm) , but theAlthough Montazer et al.  developeThere are many methods to prepare antibacterial fibers by Ag deposition, which have been extensively summarized in recent review articles ,62,63, iAlthough Ag NP coatings have been used for air conditioner filters, there are limited data on their durability. At the same time, our recent studies of AgCu NPs indicated that they showed excellent antibacterial efficacy, which can reduce Ag consumption if they meet the same antibacterial efficacy. Our intention here is focused on evaluating the durability of AgCu NP coatings for nonwoven fabrics as air filters. We also explored their advantages, comparing Ag NPs and Ag ions with PVP-PVA stabilizers, coated onto nonwoven fabric, without using any surface modification processes or special binders. We found that AgCu NP-coated fabrics showed the highest durability, while Ag ion-coated fabrics, with and without PVP-PVA stabilizers, showed the poorest.Firstly, we checked the appearance of the coated nonwoven fabrics as deposited and after running for various periods (0–30 days). After running as depicted in S. aureus are better for both AgCu NPs and Ag NPs compared to Ag ion-coated fibers. E. coli bacteria at the initial stage. However, after running for half a month, little antibacterial efficacy remained for the Ag ion-coated fabric, regardless of whether it was coated with PVP-PVA or not for both bacterial strains. For Ag NP-coated fabrics, there was an increase in antibacterial efficacy after two weeks, followed by a decrease after one month of running. In contrast, for the AgCu NP-coated fabric, the antibacterial efficacy increased after running for both a half and a full month. AgCu NP-coated fabric had the best antibacterial efficacy and durability, followed by Ag NPs, while the worst cases were Ag ion-coated fabrics, with or without stabilizers.5/2 peak is located at 367.5 eV for the as-prepared sample, while the Ag-O (or -OH) peak appears at 369 eV after running for one month. Cu2+ is seen to exist initially, as seen from the presence of the shakeup satellite peak [ite peak , with liite peak ,72.Detailed XPS spectra are shown in These color changes and analytic data  clearly show that the initial color of all coated nonwoven fabrics was changed, especially for the AgCu NP-coated fabrics, which look like uncoated fabrics. Both Ag and AgCu NP-coated fabrics showed no loss of Ag after running for one month, while Ag ion-coated samples, with or without PVP/PVA, showed some loss of Ag.The color changes in both uncoated and Ag-coated fabrics, induced by air currents in the air conditioner, as shown in Surface chemical analysis by XPS, as shown in 3, in the absence of added reducing agents, undergo a reduction reaction from Ag ions to metallic Ag (Ag+ to Ag0) [The XPS results presented in  to Ag0)  due to t to Ag0) ,80, whicHowever, the loss of Ag concentration is minimal for Ag and AgCu NPs, implying that they have a stronger interaction with the fabrics when the wind blows. This enhanced interaction is attributed to the presence of PVP-PVA that can form hydrogen bonds with the fibers . TherefoS. aureus than for E. coli. This is different than the case of Ag and AgCu NPs and Ag ions in aqueous solutions. Secondly, the antibacterial efficacy of Ag NPs is increased in the first 15-day running period, which then decreases after running for one month. The increased antibacterial efficacy of Ag NP-coated fabrics for the first 15 days of airflow may be caused by NP surface oxidation layer formation during that time. The decreased antibacterial efficacy of Ag NP-coated fabrics can be attributed to the Ag NPs aggregation; this is consistent with the Ag NP color change. As one can see in For the antibacterial efficacy change shown in It Is well known that antibacterial efficacy is dependent on the Ag NPs’ size ,83,84,85Based on this analysis, a schematic diagram for the coated fiber color and property changes is found in This study provides us with a facile and cost-effective method to maintain stable AgCu NP-based coated antibacterial nonwoven fabric, which can be considered an excellent substitute for colorless antibacterial filters applied in air conditioning to achieve air purification for human health.3, 99.8%) was obtained from Tongboxin Hongyin Products Co., Ltd. .Commercially nonwoven K5310 fabric, composed of polypropylene/polyethylene (PP/PE), was purchased from Jiangsu Beihu New Material Co., Ltd. ; Ag and AgCu NP aqueous dispersions were provided by Solmont Technology Wuxi Co., Ltd.  at 1000 ppm Ag concentration and PVP-PVA as stabilizers. Ag nitrate  and soaked in the different antibacterial agents for 1 min before the excess liquid was rolled out and the fabrics were dried by atmospheric exposure. The Ag-coated nonwoven fabrics were dried for 24 h in air at room temperature.Air blowing test: samples of fabric were glued to the air inlet of an air conditioner KFR-35GW/K150+N3, Chigo Air Conditioning Co., Ltd.,  using double-sided adhesive tape. The air conditioner was working continuously for 30 days, and samples were evaluated on days 0, 15, and 30.XPS was conducted on an ESCALab 230i, whose X-ray source was monochromatic Al Kα (1486.7 eV). Survey spectra were conducted with 1.0 eV steps at 100 eV pass energy, while high-resolution spectra were conducted with 0.05 eV steps and a 25-eV pass energy. All spectra were calibrated by placing the C1s peak for C-C/C-H at 284.8 eV.Escherichia coli (ATCC 8099) and Gram-positive Staphylococcus aureus (ATCC 6538). The sub-culture of the bacterial colony was made from 3–5 generations of the primary culture. Bacteria were grown overnight on a nutrient-agar media plate. Inoculums of 0.5 McFarland standards (1.5 × 108 CFU/mL) were maintained in nutrient broth by picking up a single colony from the sub-culture plate [6 CFU/mL for testing. Fabric samples were cut with a 14 mm punch.The antibacterial efficacy of the fabrics was evaluated against Gram-negative re plate , and fifAgar dilution is considered to be the gold standard of susceptibility testing or the most accurate way to measure the resistance of bacteria to antibiotics . In thisp < 0.05 for significantly various groups.Statistical data (average ± SD) analyses were conducted, applying One-Way ANOVA (SPSS software Version 8.0 program). This study considered The durability of directly deposited Ag and AgCu NPs and Ag ions, by dip-roll processes onto nonwoven fabric for air conditioner applications, has been evaluated by antibacterial efficacy, color change, and XPS analysis. We found that the disappearance of the yellow color of AgCu NP-coated fabrics on air current exposure is attributed to the surface oxidation of AgCu NPs without the degeneration of antibacterial activity, while the decreased antibacterial activity and color change for the Ag NP- and Ag ion-coated fabrics can be attributed to surface Ag NP aggregation and Ag loss. PVP-PVA stabilized AgCu NPs, deposited onto the fabric by dip-rolling, appear to be applicable as air conditioning antibacterial filters, leading to higher durability and enhanced antibacterial efficacy. Overall, this study proposes a facile and inexpensive method to maintain stable NP-coated fabrics without using any surface modification processes or special binders but with the improvement of antimicrobial efficacy in use, which may be an effective solution to the implementation of colorless anti-bacterial filters applied in air conditioners to achieve better air purification, particularly for respiratory health.