An aggressive lowering of blood pressure (BP) could lead to neurological worsening, particularly of the area that has not been reperfused in acute stroke patients with large vessel occlusion (LVO). We sought to investigate the association of reperfusion status and BP course following mechanical thrombectomy (MT) with outcomes in LVO.Consecutive patients with LVO treated with MT between Jan 2020 to Jun 2021 were enrolled in a retrospective cohort study. Hourly systolic BP (SBP) and diastolic BP (DBP) were recorded for 72 h following MT and maximum SBP and DBP levels were identified. The Extended Thrombolysis in Cerebral Infarction (eTICI) scale was used to assess reperfusion extent. LVO patients were stratified in 2 groups based on reperfusion status: complete reperfusion (eTICI 3) and incomplete reperfusion (eTICI 2b/c). Three-month functional independence was defined as a modified Rankin Scale score of 0–2.p = 0.006), higher likelihood of in-hospital mortality and 3-month mortality . No statistical correlation was found between reperfusion status and blood pressure level (p > 0.05). In patients with complete reperfusion, patients with an average BP 120-140 mmHg tends to have worse functional outcome compared with 100-120 mmHg .A total of 263 acute ischemic stroke patients with LVO were retrospectively evaluated. Complete reperfusion was achieved in 210 patients (79.8%). Post-MT maximum SBP over 160 mmHg was significantly related to worse functional outcome (38.1% vs. 55.7%, High maximum SBP levels following MT are associated with an increased likelihood of 3-month functional dependence and mortality. An average BP of 100–120 mmHg tends to have better functional independence in completely reperfused patients. The effect of intensive BP control on incomplete reperfusion still warrants further investigations. According to previous studies, increased systolic blood pressure (SBP) after endovascular treatment (EVT) in patients with acute ischemic stroke (AIS) has been correlated with worse clinical outcomes and an increased risk of intracranial hemorrhage (ICH) . The mosFrom January 2020 to June 2021, we enrolled consecutive patients who presented AIS within 24 h of large vessel occlusion (LVO) and underwent MT in a comprehensive stroke center. The exclusion criteria for patients were as follows: patients who had unsuccessful reperfusion status, eTICI = 0–2a; those with insufficient BP data; patients with unfavorable previous functional status, previously modified Rankin scale (mRS) score of >2; and patients who were unable to be followed-up. The process of inclusion and exclusion is shown in For evaluating the reperfusion status, eTICI  was usedThis study was approved by the Ethics Committee Boards, and the requirement for written consent was waived. All patients received verbal and written information on the collection of observational data regarding this clinical study and were free to withdraw.Preprocedural variable data of patients were collected from a prospectively defined clinical registry, including age, sex, comorbidities, including hypertension, diabetes mellitus, and atrial fibrillation, baseline National Institutes of Health Stroke Scale (NIHSS), admission NIHSS, admission Alberta Stroke Program Early Computed Tomography (CT) Score (ASPECTS), onset to reperfusion time (h), stroke cause according to the trial of ORG 10172 in acute stroke treatment (TOAST) criteria, and if the intravenous tissue-type plasminogen activator was administered. The primary clinical outcome was the 3-month functional independence (mRS = 0–2). The mRS was determined through in-person interviews in the follow-up clinics or telephonic interviews. Secondary outcomes included symptomatic ICH (sICH), asymptomatic ICH (aICH), and all-cause 90-day mortality. sICH was defined as any intraparenchymal, subarachnoid, or intraventricular hemorrhage on postprocedural CT associated with a ≥ 4-point increase in the NIHSS score according to the “European cooperative acute stroke study” criteria. Early neurological deterioration (END) was defined as a ≥ 4-point increase in the NIHSS score at 24 h compared with the baseline NIHSS.Patients were admitted to the neurointensive care unit (NICU) after EVT. Their BP was monitored every 15 min before the target was achieved, and then every 1 h, with a BP cuff for 72 h. After discharge from the NICU, the BP of patients was monitored four times a day. A standardized BP measurement protocol was enacted. The BP level was decided according to the preference of the physician, with a preponderant target of <120 mmHg, 130 mmHg, or 140 mmHg, and was maintained for at least 72 h. However, because this was a real-world analysis, while not a per-protocol analysis, namely, the BP level was not precisely equal to the BP target. Intravenous BP lowering protocols guided the titration of the locally available drugs, including urapidil, nicardipine, and nitroglycerin, through repeated bolus or infusions to achieve the BP target. An SBP of <90 mmHg was set as the threshold for anti-hypertensive medication cessation and the use of intravenous fluids and inotropes, as required.2 test, and that for the continuous variables was performed using the unpaired t-test. Furthermore, the Mann–Whitney U test, one-way analysis of variance, or Kruskal–Wallis test were performed per the requirement. Univariate and multivariable logistic regression analyses were used to evaluate the association between BP level at 27 h with the clinical outcomes, including END, sICH, aICH, in-hospital mortality, 3-month mortality, and 3-month functional independence after adjusting for potential confounders . The associations are presented as odds ratios (ORs) with corresponding 95% CI. Statistical significance was accepted at the p-value of <0.05 in the multivariable logistic regression analysis. The Statistical Package for Social Science, version 22.0 for Windows  was used for statistical analyses.Continuous variables are presented as mean ± standard deviation  and as median with interquartile range (IQR) (skewed distribution). Categorical variables are presented as percentages with their corresponding 95% confidence interval (CI). Statistical comparisons for the categorical variables between the two groups were performed using the χp = 0.011), which was similar to the proportion of patients with ICA occlusion . The risk of sICH in the complete reperfusion group (5.8%) was similar, without any statistically significant difference, to that in the incomplete reperfusion group (5.7%) (p = 1.000). Similarly, END occurred in 13.8 and 18.9% of patients in the two groups, with no statistical significance (p = 0.586). However, the risk of aICH was significantly higher in the incomplete reperfusion group (15.5%) than in the complete reperfusion group (28.3%) (p = 0.031). The proportion of patients with 3-month functional independence tend to be higher in the complete reperfusion group (50.5%) than in the incomplete reperfusion group (36.7%). Nevertheless, the proportion of patients with 3-month mortality tend to be lower in the complete reperfusion group (19.1%) than in the incomplete reperfusion group (24.5%).During the study period, 428 patients with LVO were treated with MT . The enrp = 0.381; DBP: 86.7 ± 18.3 mmHg versus 85.1 ± 16.7 mmHg, p = 0.489). In patients with a maximum SBP of >160 mmHg, the risk of sICH significantly increased . The risk of aICH was similar . END observation in both groups was comparable in patients with a maximum SBP of >160 mmHg . However, in-hospital and 3-month mortality rates were significantly higher . The 3-month functional independence was significantly higher in patients with a maximum SBP of <160 mmHg .The comparison of the baseline characteristics and outcomes in the two groups of post-MT maximum SBP among patients with successful reperfusion after EVT is presented in p > 0.05) (p = 0.061). Additionally, no linear relationship was found between post-MT SBP and clinical outcomes based on the reperfusion status (p > 0.05)  . Moreove > 0.05) .This study indicated that a maximum SBP level of >160 mmHg during the first post-MT 72 h is related to a higher likelihood of worse clinical outcomes, including in-hospital mortality, END, 3-month mortality, and 3-month functional dependence. Multivariate regression analysis did not reveal any relationship between the reperfusion status and BP level on the clinical outcomes. Additionally, patients with complete reperfusion tended to have the best functional outcome with an average SBP of 100–120 mmHg. The main finding of this study is that the association between the post-MT BP target and clinical outcome is comparable with the previous studies .ENCHANTED-2/MT is a multicenter, open-label, blinded-endpoint, randomized controlled trial that compares the safety and efficacy of more intensive BP lowering treatment (<120 mmHg) with less intensive treatment targets (140–180 mmHg) in patients with increased BP after reperfusion with EVT . The likThe AURORA meta-analysis of thrombectomy for anterior circulation stroke >6 h after the last known well revealed that the post-MT functional independence was 45.9%, with a mortality rate of 16.5% and an sICH rate of 5.3% . Herein,n = 30, 52%) and individualized on a case-by-case basis  in most institutions. Among them, 36% of physicians preferred an SBP target in the 120–139 mmHg range and 5% preferred that of <120 mmHg. In another online survey endorsed by the European Society of Intensive Care Medicine (In a study, considerable discrepancy regarding BP target was reported among different institutions and physicians . An onliMedicine , 54% of There are some limitations of this study. Because of the retrospective nature of this study, it was subjected to the biases inherent to this type of analysis. The BP levels were decided according to the preference of the physician, and not based on randomization. Furthermore, we could not evaluate the hypothesis that if BP target affected clinical prognosis, considering the information regarding the type of BP target was not adequate in the study cohort. Moreover, we did not measure the final infarct volume; therefore, we could not evaluate its association with BP level. Instead, END, which partially indicates the deterioration of cerebral infarction, was evaluated. There is also a higher proportion of patients with cardioembolic stroke in the incomplete reperfusion group, which may lead to bias. According to previous immunohistochemical staining study, platelet-rich thrombi were associated with a smaller prevalence of TICI 3 compared to platelet-poor thrombi, which is in coincidence with our result . FinallyOur study showed the preliminary data indicating that SBP of >160 mmHg, following MT, was associated with an increased likelihood of 3-month mortality and functional dependence in patients with LVO. An average SBP between 100–120 mmHg tended to have a better 3-month functional independence in patients with complete reperfusion, whereas patients with incomplete reperfusion showed worse 3-month functional independence. Further investigation is needed to determine if an incomplete reperfused area can affect the BP target.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/participants or patients/participants legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.HX, HL and PZ: drafted this manuscript. YG, HaL, HS, WH, LZ, ZL, YZ, PX, and JL: contributed in statistical analysis, data collection, and imaging analysis. XZ and PY: designed this study. All authors contributed to the article and approved the submitted version.


Cognitive impairment occurs in up to 70% of people with MS (pwMS) and has a large impact on quality of life and working capacity. As part of the development of a smartphone-app (dreaMS) for monitoring MS disease activity and progression, we assessed the feasibility and acceptance of using cognitive games as assessment tools for cognitive domains.We integrated ten cognitive games in the dreaMS app. Participants were asked to play these games twice a week for 5 weeks. All subjects underwent a battery of established neuropsychological tests. User feedback on acceptance was obtained via a five-point Likert-scale questionnaire. We correlated game performance measures with predetermined reference tests (Spearman’s rho) and analyzed differences between pwMS and Healthy Controls .rs|= 0.34–0.77). Performance improved in both groups over the 5 weeks. Average ratings for overall impression and meaningfulness were 4.6 (range 4.2–4.9) and 4.7 (range 4.5–4.8), respectively.We included 31 pwMS  and 31 age- and sex-matched HC. All but one game showed moderate–strong correlations with their reference tests, (|Moderate–strong correlations with reference tests suggest that adaptive cognitive games may be used as measures of cognitive domains. The practice effects observed suggest that game-derived measures may capture change over time. All games were perceived as enjoyable and meaningful, features crucial for long-term adherence. Our results encourage further validation of adaptive cognitive games as monitoring tools for cognition in larger studies of longer duration.ClinicalTrials.gov: NCT04413032.The online version contains supplementary material available at 10.1007/s00415-023-11671-9. Multiple Sclerosis (MS) a chronic inflammatory and degenerative autoimmune disease of the central nervous system affecting approximately 2.4 million people worldwide is one of the most prevalent neurological causes of permanent disability in young adults , 2. Up tIn recent years, there have been many advances in digitization of cognitive assessment using the potential of digital devices such as computers, tablets, and smartphones –14. EspeIn this proof-of-concept study, we correlate measures derived from adaptive smartphone-based cognitive training games with results of predefined corresponding established cognitive paper and pencil tests. The primary objective is to identify cognitive game measures that correlate with established neuropsychological tests and identify games that are well-accepted and meaningful for pwMS. Additional exploratory objectives were (a) investigating correlations of all reference test scores with all game measure scores; (b) determining whether the games and tests could be attributed to specific cognitive domains, and c) analyzing differences between pwMS and HC regarding game performance and game ratings.The Research Center for Clinical Neuroimmunology and Neuroscience Basel (RC2NB) is currently developing a smartphone app „dreaMS “, in cooperation with the medical device software manufacturer Healios AG , 18. ThiHere, we report on the performance of these cognitive games as measures of cognitive domains including their correlation with established neuropsychological tests, acceptance by users, and assessment of their meaningfulness by pwMS.This study was approved by the local ethics committee , Basel, Switzerland, on July 17th 2020/project-ID 2020-01515). All participants gave their written informed consent. This study conforms with World Medical Association Declaration of Helsinki and was registered at ClinicalTrials.gov: NCT04413032.Participants attended three visits, all of them taking place at the MS Center at the University Hospital Basel: 1. Screening visit (S), 2. Baseline visit (BL) and 3. End-of-study visit (EoS). A written informed consent was obtained from all participants at the screening visit. At BL, all participants underwent a neurological examination and completed a battery of established standardized neuropsychological tests  according to the revised McDonald criteria (2017)  and an ENumber of correct answers within a given time frame but also number of errors per se. Depending on the structure of a cognitive game additional features like qualitative and temporal patterns of errors might also be derived or a combination of such features. In adaptive games, the level of difficulty reached by the participant could also by itself be a valid measure. We prospectively assigned each game to a cognitive domain according to information provided by the game developer and expert consensus. Every Peak game was intended to train a specific cognitive domain, which was developed under supervision of certified neuropsychologists at Peak [the number of correct answers in a given time interval, since it includes both speed and accuracy. However, the games have the additional feature of difficulty-adaptation, according to a scoring system based on the number of correct answers. Since the difficulty level adapts, the measure number of correct answers can be misleading for some games. In these cases, taking the change in difficulty level itself as the measure was the preferred option .10 cognitive training games from Peak were included in the dreaMS app via deep link . Game se at Peak . To ensu at Peak . RegardiAs a general screening test of cognitive impairment we used the MUSIC (Multiple Sclerosis Inventory of Cognition) . The cogAt end of study (EoS), all participants were asked to provide feedback. For each game, participants rated four questions on a five-point Likert scale. The questions addressed the participant’s overall impression, whether the game was appropriately challenging , whether the participant would be willing to play the games regularly in the future, and whether they thought the games were relevant for MS (meaningfulness). The question about meaningfulness was only asked to pwMS. Mean ratings of those four questions were used to calculate overall acceptance.rs ≥ 0.3) correlation coefficient [The primary objective was to identify cognitive game measures that correlated with established neuropsychological tests and identify games that are well-accepted and meaningful for pwMS. Hence, the primary study outcomes were (a) correlation coefficient between game measures and the corresponding reference test scores. The average of the game measures over all ten sessions (2x/week for 5 weeks) was correlated with the corresponding established neuropsychological reference test score using spearman’s rank correlation coefficient. As an acceptable threshold, we targeted an at least moderate  useAdditional exploratory objectives were the correlations of all reference test scores (mean) with all game measure scores (mean), using spearman’s rank correlation coefficient in correlation matrices. Further, we investigated whether the games and tests could be attributed to specific cognitive domains by performing a factor analysis including all game measures (mean) and test scores (mean), using maximum likelihood estimation and the rotation method “varimax”. Differences between pwMS and HC regarding game performance were assessed by means of rank biserial correlations between the average game scores over all ten sessions and the group variable. Since the analysis of differences between the two groups was exploratory, we used the more conservative analysis method: rank biserial correlations, in order to avoid misinterpretation. Spaghetti plots of all participants’ game performances over the ten sessions were created to better visualize group differences over time. Further, we compared the mean Likert scale game ratings across pwMS and HC using rank biserial correlation.Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS).Version 28.0 and R version 4.2.0 . The study protocol and statistical analysis plan are available as supplementary material (S3: Study protocol).Ludwig Kappos, Silvan Pless, and Andrea Wiencierz take full responsibility for the data, the analyses and interpretation, and the conduct of the research, have full access to all of the data and have the right to publish any data separate and apart from any sponsor. The data supporting the findings of this study are available from the corresponding author upon reasonable request.Between October 5th 2020 and February 28th 2021, we recruited 31 pwMS from the MS Center, University Hospital Basel with a mean age of 43.4 ± 12.0 years, 68% females with a median Expanded Disability Status Scale score of 3.0 (range 1.0–6.0) and 31 age- and sex-matched HC. Table rrb|= − 0.24, p = 0.07). We did not find a strong correlation between the MUSIC and the EDSS .According to the results of MUSIC cognitive impairment (CI) was present in 7/30 (22.5%) pwMS (3 (9.7%) moderate and 4 (12.9%) mild). No CI was observed in the HC group. When comparing the mean MUSIC score between pwMS (24 ± 4.9) and HC (26 ± 3.9), no clear differences were found  reached the preset level of moderate spearman correlation coefficients (|rs|≥ 0.3) with their predefined reference tests. Six games , Spin Cycle , Rush Back , Face Switch , Perilous Path , and Low Pop  correlated strongly with their reference tests. Three , Baggage Claim , and Puzzle Blox ) showed moderate correlation coefficients. An overview of the correlation coefficients, their 95% confidence intervals and p-values is shown in Table All but the language-game  |rs|≥ 0. with theoverall impression, 4.40 (range 4.13–4.58) for perceived difficulty, 4.50 (range 4.15–4.77) for willingness to use in the future and 4.69 (range: 4.52–4.84) for meaningfulness for pwMS. All categories in all games met the predefined target of ≥ 3. Table Over all games, the mean Likert scale rating was 4.63 (range 4.15–4.9) for rs|< 0.1), 17 weak (|rs|≥ 0.1), 28 moderate (|rs|≥ 0.3), and 23 strong (|rs|≥ 0.5) correlations as shown in Table SDMT showed equal factor loadings for two factors (factor 1 and 3). Attributing every game and test to the factor with its highest factor loading, factor 1 includes 2 tests (SDMT and VLMT), and 3 games . One Test  and 5 games  showed the highest factor loadings with factor 2. Four tests: ROCF copy, Stroop, TMT b, SDMT, and the game Word Hunt had the highest association with factor 3. Lastly, the test RWT and the game Low Pop showed the highest factor loadings with factor 4 (Table Of the 70 possible correlations (10 cognitive games * 7 reference tests), we found 2 near zero (| |rs|≥ 0. correlat |rs|≥ 0. correlat 4 Table .Table 5Cn = 30) and HC (n = 29) were found: Low Pop , Baggage Claim , Face Switch , Must Sort , Perilous Path , Puzzle Blox , Rush Back , Spin Cycle , Word Hunt , Zap Gap . When the average scores of each session for each participant are plotted, an increase in game-scores over the 5-week study period is visible, with the control-group showing steeper increases in some, and higher scores and or difficulty levels reached in most games  showed a continuous increase over the ten sessions. When we plotted the data obtained from these two games using mean difficulty level instead of number of successful rounds and completion time as game measures, an increase of average performance was visible as well , no clear differences between pwMS  by group and topic (HC vs pwMS)).rs ≥ 0.3) supporting their utility as cognitive assessment tools. The fact that the language-game Word Hunt did not reach a correlation coefficient of rs ≥ 0.3 with RWT may be the effect of different assessment approaches: whereas Word Hunt relies on visual detection of words in a letter grid, the RWT is a purely verbal semantic fluency test. The overall correlation matrix, that shows moderate correlations of Word Hunt with all other reference tests (range rs = 0.36–0.69), most of which include visual tasks, supports this assumption. Similarly, the only other predefined game-test correlation below rs = 0.4: Zap Gap – Stroop, might have also been affected by the issue of different assessment approaches: while Stroop is a purely verbal test, in Zap Gap small orbs have to be touched at the right moment. Ergo visual-timing and dexterity might have influenced the comparison. Of course, other games also include a dexterity aspect which does usually not play a role in most paper pencil tests. However, dexterity required in the other games are very basic (touching large buttons to respond by yes/no). When omitting these two tests due to this effect, the other 8/10 correlations, where assessment methods are more similar, are distributed with a rather small range (rs = 0.44–0.77).All but one of the adaptive cognitive games showed a moderate–strong correlation with their respective pre-assigned established reference tests and met the predefined correlation level , and Word Hunt (language), we did not observe a clear increase in performance. For these two games, the predefined measures were raw scores (number of correct answers and completion time). We assume that the scores of these games stagnated or declined because the level-, and therefore difficulty change interfered with the measurement of raw scores. As performance improved, the difficulty level of these adaptive games increased, leading to less increase or even relative decrease in the raw scores. When we measured the performance over time by the difficulty level reached, a continuous increase in performance was seen for these two games as well despite the lower number of possible levels  and Wiley et al. (2020) also describe advantages of gamification elements regarding motivation, positive affect, enjoyment, felt challenge, meaning, and even performance in cognitive tasks , 35. SucSince this was a sub study of the dreaMS feasibility study, the sample size, schedule, and prevalence of cognitive impairment were not specifically set up for the assessment of cognitive games . The widIn this feasibility study, we demonstrate that smartphone games can provide reliable measures of cognitive function both in pwMS and HC. Although most game-derived measures correlated with their established cognitive reference tests, domain-affinity needs to be further explored in larger and more diverse populations. Practice effects were clearly depicted in both HCs and pwMS, suggesting that cognitive game measurements are sensitive to change over time and learning curves have to be taken into account in data analysis. All participants found the games appealing and meaningful and were motivated to use such a monitoring tool on a regular basis for longer periods. Further studies with longer duration in larger populations are warranted to validate such cognitive games as monitoring tools of cognition in pwMS. While the novel method explored in this study focused on monitoring disease evolution in pwMS, we acknowledge the great potential it shows as a rehabilitation tool in both MS and other medical fields.Supplementary file1 (PDF 144 KB)Supplementary file2 (PDF 85 KB)Supplementary file3 (PDF 115 KB)Supplementary file4 (PDF 7147 KB)Supplementary file5 (PDF 101 KB)Supplementary file6 (PDF 118 KB)Below is the link to the electronic supplementary material.


We used the bivariate causal mixture model (MiXeR) to estimate the total number of shared genetic variants, local analysis of co-variant annotation (LAVA) to estimate local genetic correlations, conditional false discovery rate (cond/conjFDR) to identify specific overlapping loci. The MiXeR analyses showed that 12.7k genetic variants are associated with ASD, of which 12.0k variants are shared with EDU, and 11.1k are shared with INT with both positive and negative relationships within overlapping variants. The majority (59–68%) of estimated shared loci have concordant effect directions, with a positive, albeit modest, genetic correlation between ASD and EDU  and INT . We discovered 43 loci jointly associated with ASD and cognitive traits (conjFDR<0.05), of which 27 were novel for ASD. Functional analysis revealed significant differential expression of candidate genes in the cerebellum and frontal cortex. To conclude, we quantified the genetic architecture shared between ASD and cognitive traits, demonstrated mixed effect directions, and identified the associated genetic loci and molecular pathways. The findings suggest that common genetic risk factors for ASD can underlie both better and worse cognitive functioning across the ASD spectrum, with different underlying biology.Autism spectrum disorder (ASD) is a highly heritable condition with a large variation in cognitive function. Here we investigated the shared genetic architecture between cognitive traits (intelligence (INT) and educational attainment (EDU)), and risk loci jointly associated with ASD and the cognitive traits. We analyzed data from genome-wide association studies (GWAS) of INT ( Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by difficulties in social communication and interaction as well as restrictive, repetitive patterns of behavior, interest or activities . Recent n = 18,381 cases and n = 27,969 controls and identified five genome-wide-significant loci [The pathogenesis of ASD is considered to originate from complex interactions between environmental  and geneant loci . By leveant loci . Howeverant loci . Meanwhiant loci  and showant loci .Intelligence and EDU are highly heritable traits which are major determinants of human health and well-being , 15. Furg) between ASD and cognitive abilities [g = 0.2–0.3 [g, between ASD and high INT, there are likely variants with an opposite effect on ASD and INT as well.Recent studies suggest that 35% of ASD patients have an intellectual disability . Among tbilities , 25, wit 0.2–0.3 , 26. Thi 0.2–0.3 , 28 and  0.2–0.3 . Thus, dWe have previously reported large polygenic overlaps despite low genetic correlation in mental disorders such as schizophrenia, ADHD and depression –32 by usHere, we took advantage of recent large GWAS data to determine the degree of overlapping genetic architecture between ASD and cognitive traits (INT and EDU) by applying MiXeR method. Second, we identified risk loci shared between ASD and the cognitive traits using the cond/conjFDR method. Third, we applied FUMA to annotate the identified loci to determine tissue expression and molecular functions of shared risk variants for ASD and cognitive traits .p values and z-scores) for the relevant phenotypes [n = 5305) [We obtained GWAS results in the form of summary statistics  , includin = 195,653) [general intelligence factor (g-factor). In the majority of cohorts, the g-factor was based on results on 13 different cognitive tests that required verbal and mathematical reasoning  [General Intelligence was based on data from 269,867 individuals across 14 cohorts, primarily consisting of data from the UK Biobank  . These sd=20016) . The incd=20016) .http://csg.sph.umich.edu/abecasis/metal/), of 71 quality-controlled cohort-level results files. The included GWAS data are restricted to individuals of European descent.Educational attainment (EDU) is measured as the number of years of completed schooling . The GWA2snp) for each trait, based on the distribution of z-scores and detailed modeling of LD structure. Polygenicity estimates included the number of ‘causal’ variants required to explain 90% of h2snp to prevent extrapolating model parameters into variants with infinitesimally small effects. Results were presented as Venn diagrams displaying the proportion of trait-specific and shared ‘causal’ SNPs. Dice coefficient as calculated by MiXeR was used to estimate the similarity between genetic architecture of two phenotypes. Model fit was evaluated based on predicted versus observed conditional quantile-quantile (Q–Q) plots, the Akaike Information Criterion (AIC) and log-likelihood plots (Supplementary Methods). A positive AIC indicates adequate discrimination between modeled fit and the comparative model. A negative AIC indicates inadequate discrimination between modeled fit and the comparative model.We applied MiXeR v1.3  to quantp-values in the primary and conditional trait are as small as or smaller than the observed p-values. Next, the conjFDR value for each SNP was calculated as the maximum of the two condFDR values . This represents a conservative estimate of the FDR for the association between each SNP with both traits. SNPs with a condFDR <0.01 or conjFDR<0.05 were assigned statistical significance. Since the complex correlations in regions with intricate linkage disequilibrium [We next applied the conditional(cond)/conjunctional(conj)FDR method, which leverages polygenic overlap between two traits to boost statistical power to identify loci associated with a single trait (condFDR) and loci jointly associated with two traits (conjFDR) . Cross-tilibrium  can biasilibrium  overlappilibrium , 47.z scores from the respective GWAS summary statistics.We defined independent genetic loci according to the FUMA protocol . We evalg) was estimated using linkage disequilibrium score regression (LDSR) [Genome-wide genetic correlation (rn (LDSR) . Local hn (LDSR) . See Supr2 ≥ 0.6 with one of the independent significant SNPs, using FUMA SNP2GENE [We functionally annotated all candidate SNPs in the genomic loci with a conjFDR value < 0.1 having an LD SNP2GENE . We linkSNP2GENE . If the SNP2GENE   and MiXeR-estimated genetic correlation of shared variants of ρβ = 0.24 (SD = 0.01). For ASD and EDU, those values are respectively rg = 0.21  and ρβ = 0.25 (SD = 0.02). This pattern of extensive genetic overlap but weak genetic correlation is indicative of mixed effect directions, supported by the MiXeR-estimated proportion of shared ‘causal’ genetic variants with concordant effects of 0.58 for both ASD&INT and ASD&EDU.Log-likelihood plots are shown in Figs. SWe leveraged this pleiotropic enrichment using condFDR analysis and re-ranked the ASD SNPs conditional on their association with EDU or INT, and vice versa. At condFDR <0.01, there were 9 loci associated with ASD conditional on INT Table S, of whicThe conjFDR Manhattan plots are shown in Fig. Loci were either concordant or discordant as denoted by the sign of the effect, and 68% 13/19) of the shared loci between ASD and INT had concordant allelic effect directions (Table S/19 of thp < 0.05/19) in both ASD and INT and nominally significant local genetic correlation (p < 0.05) (marked with green in Table Sp < 0.05/32) local heritabilities in both ASD and EDU and significant (p < 0.05) genetic correlation between them  with significant local heritabilities  we mapped 104 genes from candidate SNPs within loci shared between ASD and INT , NFR1 (nuclear respiratory factor 1) and vitamin D receptor.Candidate genes were enrichments in 23 immune related gene sets for ASD and EDU, among them, Interleukin-2 and Interleukin-10 pathways, Macrophage Stimulating 1 (MSP1) pathway, EBNA1 anticorrelated, and development of regulatory T cells (Tregs).As seen in Tables SNCKIPSD, CCDC36, IP6K2, PRKAR2A, QRICH1, CCDC71) showed that they were enriched in pathways for inflammatory diseases and blood protein levels  are shown in Figs. Sls Fig. S.FUMA GENE2FUNC of discordant loci  we found trends of similar enrichments as the total set of discordant genes , in line with previous findings [n = 27 (63%) had concordant effect directions with INT and EDU. Thus, the main fraction of common variants shared with ASD is associated with higher INT and EDU. These variants may shed light on mechanisms underlying better cognition in ASD patients [The current findings of bidirectional genetic overlap between ASD and cognitive traits INT and EDU, as revealed with the MiXeR method, has not been shown before. The genetic overlap estimated by Dice coefficient was 0.90–0.91 which is substantial, taking into account the relatively low genetic correlation we found between ASD and INT  , 54, bipRHOA has been targeted for improved learning and memory in ASD animal models [Analyses of brain tissue expression of all candidate genes, including both concordant and discordant showed that they are significantly upregulated in two brain tissues in frontal cortex and cerebellum, which is in line with a recent meta-analysis of post-mortem studies in ASD . In recel models . As expeCKB). This seems in line with that creatine has been suggested as a cognitive enhancer [MST1, which is found in the high intelligence-pathway and plays a role in autoimmunity [Genes associated with concordant loci were enriched in a pathway for extremely high INT , and incenhancer , The conimmunity . This suimmunity  and is cimmunity , 66. Conimmunity , 68.CRHR1, KANSL1, MAPT, and WNT3. CRHR1 encodes a corticotrophin releasing hormone receptor implicated in social behavior [KANSL1 has been associated with autistic traits [MAPT encodes the tau–protein which misfolds and forms a hallmark of frontotemporal dementia and Alzheimer’s disease [WNT3 is a Wnt-signaling gene involved in neurogenesis [Discordant credible genes were enriched in three types of GWAS phenotypes, mental disorders, neurodegenerative diseases and somatic traits. Of these, the enrichment in neurodegenerative diseases as Alzheimer’s and Parkinson’s is of interest since the variants could possibly be involved in mechanisms underlying autistic regression in children and of increased risk of dementia in adults , 66. Amobehavior , 70 and behavior . KANSL1 c traits  and cognc traits . MAPT en disease . WNT3 isogenesis , as wellogenesis . It has ogenesis . TogetheA limitation of our study is that the sample of UK-biobank consists mainly of persons of European ancestries. Another limitation is that the study does not include rare pathogenic variants causing ASD, as only common variants are included in the analyses. Furthermore, the results are based on a common factor for INT, which is not exactly similar with a full IQ score. Furthermore, EDU is not purely a cognitive trait, but it is also influenced by other factors, including socioeconomic status.In conclusion, the current findings show extensive bidirectional genetic overlap between ASD and cognitive traits, with a majority of loci for ASD associated with better cognitive performance. The mixture of effect directions is in line with the large variation in cognitive abilities in ASD. Together, these findings suggest that genetic factors may underlie some of the large variation in cognitive performance in ASD, and highlight molecular mechanisms involved in the two cognitive subgroups within the ASD spectrum.Supplementary information regarding methodsSupplementary Results, Figure S1-S9 and Table 16Supplementary Results, Tables S1-S15


Fosinopril and amlodipine are commonly prescribed as first-line pharmacotherapeutic agents for pediatric hypertension, but there is a lack of comparative studies regarding the efficacy of these two drugs. We aimed to evaluate and compare the efficacy of fosinopril and amlodipine monotherapy in pediatric primary hypertension.n = 96) and the amlodipine group (n = 79). Subgroup analysis was performed to compare the efficacy of the two groups in terms of blood pressure (BP) control rates and reductions following a 4-week treatment.This was a single-center, bidirectional observational study. A total of 175 children and adolescents with primary hypertension receiving antihypertensive monotherapy from July 2020 to February 2023 were enrolled. According to antihypertensive drugs, they were divided into the fosinopril group (P > 0.05). Further subsequent subgroup analyses revealed that the reductions in SBP and DBP in the fosinopril group were significantly greater than those in the amlodipine group in patients of females and hypo-HDL-cholesterolemia (FDR adjusted P < 0.05), and there was a trend of difference, although not significant, in patients with central obesity and insulin resistance (IR) (FDR adjusted 0.05 < P ≤ 0.1). However, there were no significant differences in treatment efficacy in patients without these characteristics. Furthermore, hypertriglyceridemia did not exhibit a significant association with the difference in treatment efficacy between the two medications (FDR adjusted P > 0.05).After 4 weeks of treatment, both groups achieved significant reductions in systolic BP (SBP) and diastolic BP (DBP) by more than 18 mmHg and 6 mmHg, respectively, with BP control rates of 61.5% in the fosinopril group and 59.5% in the amlodipine group, revealing no significant differences in the antihypertensive efficacy between the two groups except for DBP control rate (FDR adjusted Fosinopril and amlodipine monotherapy were both effective in pediatric primary hypertension during a short-term follow-up. Fosinopril may be particularly effective in reducing BP in hypertensive patients of females, central obesity, IR, and hypo-HDL-cholesterolemia. These findings indicate that optimizing antihypertensive medication selection based on the individualized characteristics of children with hypertension may improve the efficacy of antihypertensive treatment. All subjects completed necessary tests, as described in our previous study  symptomatic hypertension, (b) hypertensive target organ damage, (c) hypertension stage 2, or (d) hypertension stage 1 with inadequate response to 6-month lifestyle modification. The study population was divided into two groups: the fosinopril group . Obesity was diagnosed when BMI exceeded the 95th BMI in children of the same age and sex, and when waist circumference/height >0.48 in males or 0.46 in females, central obesity was determined . The lab2.4.An initial dosage of 10 mg daily fosinopril or 5 mg daily amlodipine was prescribed to all subjects. BP was evaluated weekly, and if necessary, the dosage was adjusted from the second week of treatment until the goal BP  was achieved or the maximum dose (40 mg daily for fosinopril and 10 mg daily for amlodipine) was reached. According to previous studies and our clinical observations, 4-week treatment of fosinopril or amlodipine can lead to a stable BP level , 19. For2.5.Subgroup analyses were performed on efficacy parameters (BP control rate and BP reduction) to compare the differences between fosinopril and amlodipine for subgroups of males and females, as well as subgroups of patients with central obesity and those without. Furthermore, homeostasis model assessments of insulin resistance (HOMA-IR) score was calculated following the formula (HOMA-IR = fasting insulin (μU/ml) × FBG (mmol/L)/22.5), and subjects were classified into subgroups of IR (>3.16) and non-IR (≤3.16) in accordance with this score . Additio2.6.P-values for treatment efficacy were adjusted by false discovery rate (FDR), which was calculated according to Benjamini-Hochberg, and all P < 0.05 were considered statistically significant. All statistical analyses were done using SPSS 25.0 and GraphPad Prism 8 software.The Shapiro–Wilk test was utilized to assess the normality of the data. Mean ± standard deviation (SD) was used to express parametric continuous data, and independent t test and paired t test were employed for between- and within-group comparisons, respectively. Non-parametric data was presented as median (the interquartile range) and analyzed using the Mann–Whitney test. Categorical variables were compared using the Chi-squared test and Fisher exact test. 3.A total of 175 subjects were included in this study, including 139 males and 36 females, with age ranged 13.0 (2.0) years, of whom 136 (77.7%) were central obesity. The median initial dosages of fosinopril and amlodipine were 0.27 and 0.06 mg/kg, respectively. The baseline characteristics of the study participants are shown in P < 0.001) (P = 0.083 and 0.093), but no differences in BP and SBP control rates, except a higher DBP control rate in the fosinopril group (P > 0.05). At week 4, the median dosage of fosinopril was increased in 53 patients to 0.35 mg/kg, while increased in 30 patients to 0.09 mg/kg of amlodipine. Furthermore, considering the potential effect of fosinopril on renal function, we followed up 82 patients prescribed fosinopril and found no significant difference in eGFR levels between before and after treatment .After 4 weeks of treatment, both the two groups achieved significant SBP and DBP reductions (< 0.001) , with BPil group . After aP < 0.05), with a borderline difference in SBP control rate (FDR adjusted P = 0.056), but these differences were not statistically significant in male patients. Additionally, in patients with central obesity, fosinopril group had a tendency to a greater reduction in SBP and DBP (FDR adjusted P = 0.07)  . To furtP < 0.05). In patients with IR, the control rate of DBP was significantly higher in the fosinopril group than in the amlodipine group, and there was a trend, although not significant, difference in the reduction of SBP and DBP between the two groups (FDR adjusted P = 0.080 and 0.100). However, no significant differences on treatment efficacy were found in patients without these characteristics (FDR adjusted P > 0.05). In addition, hypertriglyceridemia did not appear to show a relationship with the difference in treatment efficacy between the two medications (FDR adjusted P > 0.05). Notably, there were no significant differences in baseline characteristics between the treatment arms in all subgroups (P > 0.05), except for the gender in patients without central obesity. All analyses are presented in Furthermore, we observed that SBP and DBP reductions in the fosinopril group were significantly greater than those of the amlodipine group in patients with hypo-HDL-cholesterolemia (FDR adjusted P > 0.05). All adverse events were resolved at the end of follow-up period. None of the other participants reported any adverse reactions.It is worth mentioning that there were no occurrences of hypotensive events or serious adverse events occurred throughout the period of treatment. 2 patients (2.4%) in the fosinopril group suffered transient dry cough with dosage of 20 mg once daily, and 1 patient (1.3%) from the amlodipine group complained of palpitation after receiving the initial dose of amlodipine with no abnormality detected on the electrocardiogram. The incidence of adverse events was not significantly different between the two groups  . SimilarRAS activation is an important mechanism for the development of hypertension in children. ACE, the main component of RAS, converts angiotensin I into angiotensin II (Ang II), which binds to its receptor to cause vasoconstriction, and release of aldosterone, resulting in a rise in BP. RAS is known to have sex differences, and endogenous sex hormones have been shown to interact with RAS, which can be upregulated by androgens and antagonized by estrogens , 29. HowIt is estimated that at least 75% of the incidence of hypertension in children is directly related to obesity. Obese patients are prone to develop hypertension, which is mainly attributed to the excessive activation of RAAS. In obesity, adipose tissue produces large amounts of AngII, and plasma RAS levels are elevated, especially those with central obesity . In addiACE1 and CYP3A5, and study on this aspect is currently ongoing. However, this study may help guide pediatricians to choose antihypertensive agents according to the different clinical characteristics of hypertensive children and was expected to improve hypertension treatment.There are several limitations in this study, including that this is a hospital-based, single-center observational study that cannot completely avoid selection bias, and further multicenter, randomized large-scale trials are necessary. The small sample size also limited the power of subgroup analyses, and future studies with large sample sizes and predefined subgroup analyses are needed to confirm or refute our findings. The antihypertensive efficacy may be affected by individual gene polymorphisms, such as 5.Fosinopril and amlodipine monotherapy were both effective in pediatric primary hypertension during a short-term follow-up. Fosinopril may be particularly effective in reducing BP in hypertensive patients of females, central obesity, insulin resistance, and hypo-HDL-cholesterolemia. These findings indicate that the optimization of antihypertensive medication selection in conjunction with the individualized characteristics of children with hypertension may improve the efficacy of antihypertensive treatment.


The aim of this study is to report an isolated pleural cryptococcosis with pleural effusion as the only manifestation, confirmed by pleural biopsy in a patient with thymoma combined with myasthenia gravis, who developed pleural effusion of unknown origin after long-term glucocorticoids and tacrolimus therapy.Pathological examination of the right pleural biopsy tissue from a patient with unexplained recurrent pleural effusion was implemented. Morphological analysis of the fungal component and metagenomic next-generation sequencing (mNGS) on the pleural tissue were performed.Cryptococcus neoformans was detected by mNGS with a species-specific read number (SSRN) of 4, confirming the diagnosis of pleural cryptococcosis. Pleural effusion was eliminated with amphotericin B and fluconazole, and healthy status was maintained at the time of review 1 year later.A biopsy specimen of the right pleura revealed numerous yeast-like organisms surrounded by mucous capsules and Cryptococcosis, manifested by simple pleural effusion, is extremely rare, but when repeated pleural effusion occurs in immunocompromised patients or in patients with malignant tumors, the possibility of cryptococcosis should be treated with high vigilance and pleural biopsy is recommended if necessary in order to confirm the diagnosis. Cryptococcus has gone from an environmental saprophyte to a global pathogen , tacrolimus capsules (10 mg twice daily), and pyridostigmine bromide tablets (60 mg twice daily) for 6 months. Five days before admission, she had a chest CT that had a little pleural effusion on the left side Figure 1According to the above examination, the neurologist thought that the chest tightness was due to the recurrence of myasthenia gravis. Methylprednisolone (40 mg/day) combined with gamma globulin (400 mg/kg) was given. After 3 days of treatment, the patient’s dyspnea worsened. A chest CT was taken again and showed a new bilateral pleural effusion Figure 16/L (61.3% multinucleated cells). The right pleural fluid was also a pale yellow exudate with the following laboratory results: protein 45.1 g/L, glucose 8.23 mmol/L, LDH 147 U/L, and cell count 1,420×106/L (58.4% multinucleated cells) , glucose 4.71 mmol/L , LDH 206 U/L , and cell count 2,223×10d cells) Table 1.The patient was referred to our respiratory department for unexplained pleural effusion and dyspnea. In view of the patient’s history, we believed that both pleural metastases and pleuro-specific infection should be considered. PETCT was performed and showed multiple pleural thickening, and metabolic activity was found bilaterally with a maximum standard uptake value of 12.5 Figure 2Cryptococcus was highly suspected by Alcian Blue–Periodic Acid-Shiff (AB-PAS), hexamine silver, and mucicarmine staining of pleural tissue (Cryptococcus neoformans was detected with a species-specific read number (SSRN) of 4 . Ultimately, a diagnosis of pleural cryptococcosis was established in this patient, although there was no exposure factor for Cryptococcus.Right pleural biopsy was performed. Pathological examination revealed fungal bodies were between mesothelial cells and histiocytes, which were numerous yeast-like organisms surrounded by mucinous capsules. l tissue Figure 3Prior to treatment, a lumbar puncture was done to rule out central nervous system infection. A therapy of amphotericin B was given. Then, the volume of pleural effusion gradually decreased and the reexamination of thoracic B ultrasound showed no pleural effusion after 1 week. Amphotericin B was discontinued after the full dose and then fluconazole was administered orally for 2 months. The follow-up chest CT revealed no pleural effusion, and the healthy status of the patient was maintained 1 year later Figure 4Cryptococcus is a rare opportunistic pathogenic fungus found in the environment in association with soil, pigeon guano, and trees. It was first described as a human pathogen by pathologist Otto Busse and surgeon Abraham Buschke in 1894 when they isolated a “Saccharomyces- like” organism from a bone infection in a young woman and was finally named Cryptococcus neoformans by Jean-Paul Vuillemin in 1901  was low (The diagnosis of cryptococcosis is graded according to probability (probable or confirmed) due to the lack of typical clinical symptoms and the variable sensitivity and specificity of radiologic and mycologic tests . Serum c was low . In our C. neoformans via either pleural fluid culture or antigen detection , accession number PRJEB64498.The data presented in the study are deposited in the ENA repository  for the publication of any potentially identifiable images or data included in this article.HJ: Data curation, Formal Analysis, Writing – original draft. HL: Formal Analysis, Project administration, Writing – original draft. MT: Data curation, Software, Writing – review & editing. YW: Formal Analysis, Investigation, Writing – review & editing. XH: Methodology, Writing – review & editing. GZ: Conceptualization, Funding acquisition, Writing – review & editing.


To describe the clinical presentation of acute dystonia (AD) from drug abuse or misuse, as well as the emergency department (ED) management and outcomes in adolescents and young adults.  This was a retrospective cohort study of patients aged 10–25 years who were admitted to the ED for AD due to intentional abuse or misuse from January 1, 2014, to June 30, 2017. Data were collected from electronic medical records by three investigators with excellent interrater reliability (0.87).  Sixty-two cases met the criteria with male predominance (85.5%); the mean age was 16.7 years. Perphenazine was the most common cause of AD (38.7%), followed by haloperidol (32.2%). The most common AD manifestations were torticollis (51.6%), oromandibular dystonia (45.2%), and oculogyric crisis (22.6%). Intravenous (IV) diazepam combined with oral trihexyphenidyl and IV diazepam alone were the most frequently used first treatment in our ED . Overall, the improvement rates from IV diazepam alone or combined with trihexyphenidyl ranged from 46.2%–75.0%. These rates were inferior to those observed with IV benztropine (100%) alone or combined with trihexyphenidyl. All patients were treated on an outpatient basis, except for one who was admitted to a pediatric ward.  In recent years, drug-induced AD caused by intentional abuse among adolescents and young adults has become a concern in Thailand. The most common suspected drugs of abuse were first-generation antipsychotics, perphenazine, and haloperidol. The most effective treatment was benztropine. During 2014-2015, we observed an unprecedented number of adolescents and young adults presenting to our emergency department (ED) with acute dystonia (AD). They often reported having tried “some pills” obtained from their peers to obtain a pleasurable effect.In clinical settings, AD often occurs as an adverse drug reaction induced by antipsychotic drugs, more frequently with high-potency typical agents, e.g., haloperidol and fluphenazine. Some of atypical agents such as olanzapine and quetiapine can also cause AD but with low incident rates . Due to Only a limited number of studies have investigated the clinical findings and ED management of drug-induced AD –8, and tThis retrospective cohort study was approved by the Ethics Committee of Vajira Hospital, and the data from January 1, 2014, to June 30, 2017, were collected from the electronic medical records of Vajira Hospital, Bangkok, Thailand. The hospital is a university hospital with 875 beds. Our emergency department has approximately 50,000 visits per year (140 visits a day). We included adolescents and young adults aged 10–25 years who were diagnosed with intentional abuse- or misuse-related AD. This age range of adolescents and young adults was defined by the Association of Maternal and Child Health Programs . The terWe collected the following data: age, sex, history of ingestion of AD-inducing drugs, laboratory test results of blood chemistry and complete blood counts, ED treatment, duration of ED and hospital stay, and ED revisit within one week after ED discharge.The characteristics of drugs reported by those patients are provided in In a few cases, urine samples were sent for confirmatory comprehensive drug screening through liquid chromatography-tandem mass spectrometry, quadrupole time-of-flight .For the treatment of each patient received, each cycle of treatment was separated by complete resolution of AD, followed by relapse. Each cycle of treatment involved monotherapy or a combination of medications; for example, the first treatment could involve the intravenous (IV) administration of diazepam, followed by oral trihexyphenidyl. We also examined the rate of ED revisit within 1 week after discharge.Data extraction was performed by three investigators using a standardized data collection form and protocol. The three investigators were trained to collect data on ten mock medical records, and then, interrater reliability (kappa) was tested from the data extracted from ten actual medical records. The kappa scores ranged from 0.83 to 0.89 (mean: 0.87), indicating excellent interrater reliability. To minimize selection bias, those three investigators were blinded to the objectives of the study, as suggested by Gearing et al. . Lastly,During the study period , 62 cases were included in this study. The number of abuse and misuse cases was 61 cases and 1 case, respectively. Among them, the majority were male . All patThe monthly average number of cases throughout the entire study period of 3.5 years was 1.5 cases. In 2015, there was a four-fold increase in the number of cases versus the average of the entire study period . Our orin = 25; 40.3%), followed by haloperidol  and promethazine . The onset of AD symptoms after drug ingestion was calculated based on data of 60 cases, yielding a mean of 27.2 ± 17.1 h. The most common drug of abuse as coingestion  was tramadol .As shown in Index case 1 (taking “B5” white tablet): haloperidol, diazepam and its metabolites , nicotine, cotinine, hydroxycotinine, tramadol, nortramadol, dinortramadol, chlorpheniramine, and norchlorpheniramineIndex case 2 (taking “B5” white tablet): haloperidol, acetaminophen, and diphenhydramineIndex case 3 (taking “white ATC”): haloperidol, cetirizine, trihexyphenidyl, caffeine, theophylline, theobromine (caffeine metabolite), diazepam and its metabolites , 7-aminoclonazepam, 7-acetamidoclonazepam, drofenine, and fenfluramineUrine samples from three patients were sent for LC/MS/MS, Q-TOF confirming the causative agents of AD as follows:Trihexyphenidyl and/or diazepam and its metabolites were from the treatment received in the ED (index cases 1 and 3).The vital signs of all cases are mostly within normal limits with a slightly elevated heart rate . Neurolon = 32; 51.6%), followed by oromandibular dystonia , and oculogyric crisis   .A total of 51 patients (81.0%) underwent blood laboratory examinations, and the results are shown in The patients were treated in the ED using one or a combination of the following medications: benztropine (2 mg intramuscularly or intravenously), diazepam (5–10 mg intravenously), and trihexyphenidyl . Interestingly, all patients treated with parenteral benztropine alone or in combination with other medications, regardless of the cycle of treatment, recovered completely without any relapses in the ED or revisited the ED within 1 week. Of note, IV diazepam alone (the most commonly used medication for drug-induced AD in our ED) in either of the three cycles of treatment was associated with success rates without relapses ranging from 46.2%–61.9%. When IV diazepam was combined with oral trihexyphenidyl, these rates ranged from 52%–75%. Two patients relapsed after receiving the third cycle of treatment; both received IV diazepam. After 13 h 11 min in the ED, one of them  was discharged. The other patient (14-year-old boy) spent 11 h 30 min in the ED and was subsequently admitted to a pediatric ward where he further received multiple doses of IV diazepam and oral trihexyphenidyl. However, he was discharged after 3 days in the hospital.The ED triage-to-discharge time and treatment-to-discharge time among those who received the first cycle of treatment were 06:10 and 05:10 h, respectively.Forty-five patients (72.6%) received trihexyphenidyl (6–12 mg/day for 1–5 days) as home medication. During the 7-day follow-up period, two cases revisited the ED due to relapse of AD. One of them  left the ED early, against medical advice, after receiving a dose of IV diazepam and showing improvement, but trihexyphenidyl was not brought back home with him. Approximately 18 h later, he returned to the ED due to focal limb dystonia. The other patient  received IV diazepam and showed improvement. He was observed in the ED for 6.5 h prior to discharge with trihexyphenidyl prescribed as home medication. However, he did not take trihexyphenidyl in the ED or at home. At 1.5 h after discharge from the ED, he returned to the ED with opisthotonos and oculogyric crisis.In the past years, antipsychotics have gained attention as potential drugs of abuse or misuse worldwide –18. MostPrevious studies on drug-induced AD from Korea , Turkey Interestingly, the physical examination data analyzed in our study did not show the antimuscarinic effects of promethazine . AlthougDiazepam (IV) was the first drug of choice in our ED since it is readily available, in addition, because diazepam (IV) and trihexyphenidyl  are covered by the universal healthcare program of the government of Thailand and are inexpensive  and trihexyphenidyl (2 mg) 0.5 Thai baht per tablet (1 USD = 32–35 Thai baht)), whereas benztropine (IV) is not, and it is relatively expensive . In general, the onset of diazepam (IV) and benztropine (IV) action occurs within a few minutes , 24, whiWe observed that IV diazepam, administered at an optimal dosage of 5–10 mg (0.1 mg/kg) for the treatment of AD , could rFinally, we tried to find risk factors for relapse; however, none of the following factors were associated with relapse either with univariate or multivariate analysis : age, seThis was a retrospective study, performed in a single center, with some missing data.This study investigated drug-induced AD from drug abuse/misuse among adolescents and young adults. Abuse was much more common than misuse. The most common suspected drugs of abuse were perphenazine and haloperidol. Relapses of AD from drug abuse during ED stay were common, particularly in patients treated with IV diazepam, oral trihexyphenidyl, or both. Benztropine was more effective; none of the patients receiving this medication had relapses.


The odds ratio for detection was 1.09 (95% CI 1.03–1.15) per 100 parts per million (ppm) increase in CO2, and 0.88 (95% CI 0.80–0.97) per stepwise increase in natural ventilation . CO2 concentration and portable air filtration were independently associated with pathogen concentration. Each 100ppm increase in CO2 was associated with a qPCR Ct value decrease of 0.08 (95% CI −0.12 to −0.04), and portable air filtration with a 0.58 (95% CI 0.25–0.91) increase. The effects of occupancy, sampling duration, mask wearing, vocalisation, temperature, humidity and mechanical ventilation were not significant. Our results support the importance of ventilation and air filtration to reduce transmission.Currently, the real-life impact of indoor climate, human behaviour, ventilation and air filtration on respiratory pathogen detection and concentration are poorly understood. This hinders the interpretability of bioaerosol quantification in indoor air to surveil respiratory pathogens and transmission risk. We tested 341 indoor air samples from 21 community settings in Belgium for 29 respiratory pathogens using qPCR. On average, 3.9 pathogens were positive per sample and 85.3% of samples tested positive for at least one. Pathogen detection and concentration varied significantly by pathogen, month, and age group in generalised linear (mixed) models and generalised estimating equations. High CO Surveillance of respiratory pathogens in air may improve understanding of indoor transmission risks but impacts of context-specific factors on pathogen abundance are not well understood. Here, the authors investigate factors associated with 29 respiratory pathogens through surveillance of 21 community settings in Belgium. Airborne transmission is almost exclusively an indoor phenomenon10. Its risk to susceptible attendants depends on pathogen, host, behavioural and environmental/building related factors10. Pathogens differ in their ability to colonise hosts and survive in the environment while retaining infectiousness12. The number of hosts, their respiratory activity, mask wearing and individual predisposition influence aerosol generation16. Environmental/building related factors such as room volume and airflow patterns, temperature, humidity, UV radiation, ventilation and air filtration may impact aerosol transport, settling, inactivation and removal11.Many respiratory infections are transmitted via the airborne route2 concentration, which reflects poor ventilation, was directly associated with school absence due to illness and with common cold symptoms17. Low air exchange rates per person through mechanical ventilation were associated with higher incidence of pneumococcal disease during a prison outbreak and with a higher risk of tuberculin conversion in healthcare workers18. The evidence to support transmission reduction by means of portable air filters—which are more affordable than pathogen removal by classical Heating, Ventilation and Air Conditioning (HVAC) systems19—is more limited. They were associated with a reduced incidence of invasive aspergillosis and reduced surface contamination with Methicillin-resistant Staphylococcus Aureus21.There is some evidence supporting the use of ventilation to reduce infectious disease incidence. High CO22. In other studies, indoor CO2 concentration was associated with higher detection of rhinovirus bioaerosols in ambient air, higher concentration of bacterial cell wall components and culturable bacterial colony forming units24. The presence of an advanced mechanical ventilation system with high-efficiency particulate absorbing (HEPA) filtration, directional flow or increased air changes per hour (ACH), correlated with lower fungal colony forming units per unit volume in hospital settings25. On the other hand, bacterial bioaerosol loads were similar across areas with mechanical, advanced mechanical and natural ventilation in the same study. Another recent study reported that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral copies were more abundant in aerosols collected in closer proximity to an infected individual placed in a controlled environment. They also correlated positively with nasopharyngeal viral copies and ambient CO2. On the other hand, they correlated inversely with ventilation, portable air filtration and increased humidity26. As for portable air filtration, experiments using particle counters showed that portable filters sped up the clearance of airborne particles28. Two small studies also suggested a reduction in detection of SARS-CoV-2 genetic material in ambient air, but the effect was not significant30. In contrast, Conway-Morris et al. (2022) did see a significant reduction in the detection, by qPCR, of SARS-CoV-2 and other respiratory pathogens31. No study has thus far performed a multivariate analysis which controlled for other important variables  when assessing the influence of either ventilation or portable air filters on the load of respiratory pathogen bioaerosols in real-life settings.The quantification of respiratory pathogens or their genetic material in indoor air has been used to study the influence of environmental factors on disease transmission. This approach has the advantage of not requiring clinical follow-up of attendants. Dinoi et al. (2022) recently combined data from 73 studies performing qPCR on indoor air samples and showed that the SARS-CoV-2 bioaerosol load was lowest in outdoor air, and higher in indoor air from hospitals than from community settings, which points at the link between pathogen detection, by qPCR, and the risk of transmission for occupants33. During the COVID-19 pandemic, pathogen detection in sewage was scaled and provided important policy insights. One benefit of environmental samples is their independence from clinical test indications, tendency for testing or laboratory capacity. Sewage sampling can surveil the population of entire cities, but also has disadvantages. Samples are highly contaminated with environmental microorganisms, runoff times may be long and variable, and—especially for respiratory pathogens—the relationship between gastrointestinal shedding and the risk of transmission may be complex34. Air sampling may be an interesting and complimentary alternative.In addition to quantifying transmission risk, sampling and testing of indoor air for respiratory pathogen bioaerosols may become an important add-on to other data sources for epidemiological surveillance, such as clinical samples, sentinel surveillance and sewage monitoring36. A recent study demonstrated the promise of multiplex qPCR on indoor air samples from community settings to track the presence of SARS-CoV-2 and other respiratory pathogens33. However, before this approach can be rolled out at scale, the factors influencing pathogen detection and concentration need better characterisation.QPCR on ambient air has long demonstrated its ability to detect pathogen presence, concentration, and genotypeWe aimed to empirically identify the host, pathogen, behavioural and environmental/building factors which correlate with a higher respiratory pathogen bioaerosol load, as assessed by qPCR, in indoor ambient air. We hypothesised that factors shown or suspected to contribute to airborne transmission would be associated with higher bioaerosol loads. If so, this would validate the use of qPCR on air samples as a proxy to quantify transmission risk and the effect of transmission reduction efforts. Also, these same factors would need consideration when performing qPCR on indoor air samples for epidemiological surveillance.2 concentration, temperature and relative humidity. See Methods for detailed definitions of each assessed variable. In an interventional sub-study, we evaluated the effect of portable air filters in a nursery. In an exploratory analysis, we investigated whether the pathogens found in ambient air samples from community settings corresponded to the pathogens found in patients with severe respiratory infections in the same region and period. We therefore retrieved the results of the same, 29 respiratory pathogen qPCR panel, performed on respiratory samples of patients at University Hospitals Leuven37.In a prospective study over a 7-month period, we therefore tested indoor ambient air from community settings in Belgium for 29 respiratory pathogens using qPCR. We investigated which of the following pathogen, host, behavioural and environmental/building related variables influenced their detection and concentration: the number of attendees, attendee density (number of attendees divided by room volume), sampling duration, mask wearing, vocalisation (voice use), natural ventilation (opening of doors and windows), portable air filtration, presence of mechanical ventilation, local COVID-19 incidence, indoor COWe collected 341 environmental air samples in 21 sampling sites between October 2021 and April 2022. See Supplementary Table Streptococcus pneumoniae (58%), human enterovirus (incl. rhinovirus) (54%), human bocavirus (45%), human adenovirus (40%) and human cytomegalovirus (38%). The percentage of samples which were positive for at least one pathogen was highest in the 3- to 6-year-old age group  followed by 0-3 years , 25–65 years , 12–18 years , 18–25 years , 6–12 years  and over 65 years . Supplementary Fig. When comparing positivity rates of all air samples, the most frequently detected pathogens, in descending order, were p 30/30, 00% follop 30/30, 00% folloStreptococcus pneumoniae were almost always positive. We observed a long peak of human adenovirus and Pneumocystis jirovecii over the winter. Other pathogens had shorter peaks, such as Human coronavirus 229E, Human coronavirus HKU-1, Human coronavirus OC43, enterovirus D68, influenza A virus, human parainfluenza virus 3, respiratory syncytial virus A/B and SARS-CoV-2. Supplementary Fig. Temporal variations in the positivity rates of pathogens are apparent in Fig. In an exploratory analysis, we assessed whether pathogen detection rates in ambient air in community settings corresponded with those detected in patients with severe respiratory infections in University Hospitals Leuven. This hospital is adjacent to the nursery and drains most patients in the region. In the nursery, the visual association was observed most clearly for SARS-CoV-2 (N positive air samples = 46) and, with much less positive samples, for enterovirus D68 and influenza A virus (N positive air samples = 4 for each) Fig. . When cop-value < 0.05). Subsequently, observations with imputed variables were removed to confirm the observed associations.We determined independent effects of a range of variables on airborne pathogen detection and concentration, by considering the qPCR result of each pathogen in a sample as a separate observation. The pathogen was considered a covariate in the resulting models, several of which included corrections for within-sample correlation. Missing data were imputed, and for each model, backward elimination was performed until only statistically significant variables remained  was associated with decreased pathogen detection. However, after exclusion of observations with imputed variables from the resulting model, vocalisation was removed as significant variable  per stepwise increase in natural ventilation . Significance levels and effect sizes were almost identical in the mixed effects logistic regression and generalised estimating equations models, both correcting for within-sample correlation , other coronaviruses, Pneumocystis jirovecii and Streptococcus pneumoniae. Contradictorily, we found a negative association with the detection of human bocavirus. As for natural ventilation, it was negatively associated with the detection of Pneumocystis jirovecii and respiratory syncytial virus A/B. Supplementary Table 2 and natural ventilation with pathogen detection.To assess whether these associations held true for pathogens individually, we used the retained independent variables from these models to run a logistic regression model with backward elimination for each pathogen. These analyses had less power due to lower sample sizes. However, a significant association remained between mean COp-values and effect sizes before backward elimination. Backward elimination on this data, which included imputed datapoints, left pathogen, month, age group, CO2 and air filtration as significant variables. Each 100ppm increase in CO2 concentration was associated with a decreased qPCR Ct value of 0.08 (95% CI 0.04 to 0.12). Natural ventilation was not significantly associated with pathogen concentration, which contrasts with the previous analysis. However, air filtration was significantly associated with pathogen concentration, with a 0.58 (95% CI 0.25 to 0.91) increase in Ct value in its presence. Significance levels and effect sizes were almost identical when excluding imputed values, or when running a linear mixed effects model correcting for within-sample correlation  of human adenovirus, human bocavirus, human cytomegalovirus, and Streptococcus pneumoniae. Contradictorily, it was associated with a lower concentration  of respiratory syncytial virus A/B. Air filtration was associated with lower concentrations of human bocavirus, human cytomegalovirus, other coronaviruses, and Streptococcus pneumoniae  installed, with a total theoretical clean air delivery rate of 999 m3/h and a resulting number of ACH of 6.1 . Location 1 had no air filtration, location 2 had three Blue PURE 221 filters (Blueair®) installed, with a total theoretical clean air delivery rate of 1770 mQ test showed no significant difference between Mondays, Wednesdays, and Fridays in location 1 (p = 0.6762). In location 2, a significant difference was present across days (p = 0.0006). Pairwise comparisons demonstrated a difference between Mondays and Wednesdays (p = 0.0229) and Mondays and Fridays (p = 0.0009) but not between Wednesdays and Fridays (p = 1). In location 3, the difference between days did not reach significance, but there was a trend (p = 0.0701).First, we compared the positivity for any respiratory pathogen between three phases of air filtration in each location separately: no ongoing filtration (Mondays), 48 h of continuous filtration (Wednesdays) and 96 h of continuous filtration (Fridays) Fig. . Cochranp = 0.9506 when comparing Mondays to Wednesdays and 0.7101 when comparing Mondays to Fridays). In location 2, there was a significant increase in Ct value of 1.22  from Mondays to Wednesdays, and 1.13 from Mondays to Fridays . In location 3, the difference in Ct value was not significant when comparing Mondays and Wednesdays . However, there was a significant increase on Fridays compared to Mondays . Supplementary Table Next, we used linear mixed effects regression models to evaluate the change in average concentration of respiratory pathogens on Wednesdays and Fridays, compared to baseline on Mondays, in each location separately. We saw no significant change in average Ct values throughout filtration phases in location 1 , this may imply that the link between variations in community circulation and morbidity was stronger for SARS-CoV-2 than for other pathogens.Both the recent study by Ramuta et al. 2022) and ours demonstrate the scalability of performing multi-pathogen qPCR on air samples from community settings to highlight pathogen presence022 and oThe current and previous studies clearly indicate that (multiplex) qPCR on ambient air from community settings can be a complementary surveillance tool to track the circulation of respiratory pathogens. However, in addition to local epidemiology, periodical differences in pathogen detection and quantity can be explained by variations in behaviour, environmental factors, technical/analytical factors, or a combination. This underscores the need to characterise the sampling sites and standardise air sampling and analysis, to interpret the epidemiological relevance of pathogen presence and concentrations in ambient air.2 concentration, after correcting for a range of confounding variables. Results were consistent across models . Natural ventilation was also negatively associated with pathogen detection, even though our models corrected for CO2 concentration. This may result from the fact that CO2 is an imperfect marker for ventilation10. These results confirm that bioaerosol load in indoor ambient air correlates strongly with low levels of ventilation , preschool (3–6 years), primary school (6–12 years), secondary school (12–18 years), adults (18+) and nursing homes (65+). See Supplementary Table 2 and humidity either manually  or using a remote climate sensor . We used the former for 58 samples and the latter for 283.We sampled for 2 h unless site-specific schedules required shorter sampling (e.g. lunch time in schools). An AerosolSense active air sampler collected air in standard AerosolSense Capture Media   from the sampler, they were transported to the lab on the day of collection. One of two sponges was lysed in transport buffer  to be used for the TaqPath qPCR assay for SARS-CoV-2. The other was lysed in Universal Transport Medium (UTM), to be used for the multiplex qPCR respiratory panel. Samples were stored at 4 degrees until processing. If they required storage over the weekend, they were frozen at −80 degrees Celsius.For the TaqPath qPCR analysis, we used the MagMAX™ Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit for automated extraction  on 200 μl sample input. For internal control, samples were spiked with a purified MS2 bacteriophage as per the manufacturer’s instructions . Extracted RNA was eluted from magnetic beads in 50 μl MagMAX Viral/Pathogen Elution Buffer.47. The elution volume of TNA was 110 µl.For the multiplex respiratory panel, Total Nucleic Acid (TNA) extraction started from 500 µl of air sample in UTM with NucliSens extraction reagents on easyMAG or eMAG . We used the specific B protocol on the instrument after off-board lysis for 10 min and continuous shaking. A 10 μL mixture of Phocine Distemper Virus and Phocine Herpesvirus was added to the lysed sample before extraction as RNA and DNA internal controls48).Extracted RNA was eluted from magnetic beads in 50 μl of UltraPure DNase/RNase free distilled water. RT-qPCR testing was performed with the TaqPath COVID-19 CE-IVD RT-PCR kit (Thermo Fisher Scientific). Results were analysed using the FastFinder analysis software  and expressed as a cycle threshold (Ct) for the ORF1ab, N, and S gene targets . The end volume of each PCR reaction mix was 20 µL: 5 µL of TNA, 5 µL of master mix  and 10 µL of primer/probe mix  and twenty-two viruses: influenza A virus, influenza B virus; human parainfluenza viruses 1 to 4; respiratory syncytial virus A/B; human enterovirus (incl. rhinovirus); enterovirus D68; herpes simplex virus type 1; herpes simplex virus type 2; Human metapneumovirus; human adenovirus; human bocavirus; human parechovirus; Human coronaviruses 229E, HKU-1, NL63 and OC43; human cytomegalovirus; Middle East respiratory syndrome coronavirus (MERS-CoV); SARS-CoV-1/2 through the ORF1ab target. Since all positive results for ORF1ab were attributed to SARS-CoV-2, rather than SARS-CoV-1, the panel could detect 22 viruses and 29 pathogens in practice. Both an RNA (Phocine Distemper Virus) and DNA internal control (Phocine Herpesvirus-1) were run with each panel47. Two internal quality control samples (Respi 3 and Respi 4) were run on alternating days with the respiratory panel. This is standard practice in clinical routine. Respi 3 contains positive material for human bocavirus, Chlamydia psittaci, Human coronaviruses NL63, 229E and HKU-1, MERS-CoV, human enterovirus (incl. rhinovirus), enterovirus D68, herpes simplex virus type 1, Human metapneumovirus, influenza A virus, human parechovirus and respiratory syncytial virus A/B. Respi 4 contains positive material for human adenovirus, Chlamydia pneumoniae, Human coronavirus OC43, SARS-CoV-1/SARS-CoV-2 Wuhan, Coxiella burnettii, human cytomegalovirus, herpes simplex virus type 2, influenza B virus, Legionella pneumophila, Mycoplasma pneumoniae, Streptococcus pneumoniae, and Pneumocystis jirovecii, human parainfluenza viruses 2 and 3, respiratory syncytial virus A/B.The panel detects seven non-viral pathogens  samples, cultures and clinical samples. The analysis was performed under ISO15189:2012 accreditation. Test results were downloaded from the University Hospitals Leuven laboratory information system as CSV files.Clinical samples which are analysed using the multiplex qPCR respiratory panel undergo the same procedure as air samples did in our study. They are transported to the laboratory in UTM immediately after collection. Lysis, storage, nucleic acid extraction and the detection of pathogens follow identical procedures.Test results were downloaded from the University Hospitals Leuven laboratory information system as CSV files.49.Weekly COVID-19 incidence Leuven: COVID-19 incidence for the city of Leuven in the seven days until the day before sampling, per 100,000 inhabitantsThe following variables were registered before and after each collected air sample:Predominant age group at the sampling site: 0–3 years, 3–6 years, 6–12 years, 12–18 years, 18–25 years, 25–65  years, +65  years. . The number of attendees was estimated by headcount both at the start and end of sampling and averaged per sample.Attendee density: averaged number of attendees divided by sampling room volume  at the start and end of sampling. Average per sample.Vocalisation: estimated by Likert scale  at the start and end of sampling. Average per sample.Natural ventilation: estimated by Likert scale  at the start and end of sampling. Average per sample.Air filtration: binary , manual entry per sample.Mechanical ventilation: binary (absent/present), manual entry per site. See Supplementary Tables 1, 7.2 concentration: numeric (parts per million/ppm). Either measured manually at the start and end of each sample and averaged or measured continuously and averaged over the total sampling duration (<11 min before start of sampling until <11 min after end of sampling).Indoor COIndoor temperature: numeric . Either measured manually at the start and end of each sample and averaged or measured continuously and averaged over the total sampling duration (<11 min before start of sampling until <11 min after end of sampling).Relative humidity: numeric (%). Either measured manually at the start and end of each sample and averaged or measured continuously and averaged over the total sampling duration (<11 min before start of sampling until <11 min after end of sampling).Manual data collection took place on paper, after which it was inputted in Excel version 16.68 (Microsoft®). Continuous measurements of ambient air parameters were collected on the web based platform  and downloaded as CSV files.To test the effectiveness of portable air filters to reduce bioaerosol load, we placed them in two separate locations in a nursery (Locations 2 and 3). Another separate space was the control (Location 1). The same group of up to 20 toddlers and 1 to 4 caregivers occupied each location during sampling. No one was present over the weekends. Supplementary Fig. 3/h. From January 17 onwards, three devices were present in nursery location 2. On the first 7 days of air filtration in this location, the air was sampled without filtration, filtered for several hours, then sampled again with active filtration. From February 7 onwards, three Philips 3000i (Philips®) devices were additionally placed in location 3 . This is another HEPA and carbon filter-based device with a clean air delivery rate of at least 333 m3/h when operated in “turbo” mode as per manufacturer specifications. The devices were used in stage 2, which corresponds to a CADR of 186.7 m3/h per device. From this moment onwards, sampling took place concurrently in all three locations. On Mondays, air filtration started after the completion of 2 h of sampling. Air filtration then continued uninterrupted for 96 h. On Wednesdays and Fridays, sampling was repeated in each location, again for 2 h per day. Air filtration was discontinued after sampling on Fridays.The study assessed two types of air filters. The Blue PURE 221 (Blueair®) is a HEPA and carbon filter-based device with a clean air delivery rate of 590 mWhen describing pathogen detection patterns across sampling sites, age groups, and time, we considered each of the 29 target pathogens separately.th due to financial constraints. The TaqPath SARS-CoV-2 result was missing for one sample and the respiratory panel for two samples due to failed transport between labs.Only the TaqPath SARS-CoV-2 qPCR was considered for SARS-CoV-2, to avoid duplication and because it is more sensitive  in all models to estimate effect sizes of the most important variables. 95% confidence intervals were computed as follows: coefficient estimate ± standard error * 1.96. We used the Wald test to estimate p-values in generalised estimating equations models and the Chi squared test for (mixed effects) linear and logistic regression models. P-values were not corrected for multiple hypothesis testing. After backward elimination, we removed observations with imputed variables to confirm the results.After imputing missing variables, as described in In an exploratory analysis, we evaluated whether the influence of variables found to be significant in the above models differed by pathogen. We ran logistic and linear regression models with, respectively, pathogen detection and Ct value as outcomes. Models were run for each detected pathogen separately, only using the retained significant variables from the models including all pathogens.p-values were obtained using the Kenward–Roger approximation of the T-distribution (pbkrtest package in R50).Lastly, we evaluated the effectiveness of portable air filtration by focusing on repeated samples taken in the three nursery locations. We used a Cochran’s Q test to compare pathogen detection rates between three phases of air filtration for each location separately: no ongoing filtration (Mondays), 48 h of continuous filtration (Wednesdays) and 96 h of continuous filtration (Fridays). Pairwise Cochran’s Q tests followed when the difference in phases was significant. We used Holm correction for multiple testing. We used mixed effects linear regression models to evaluate the effect of different air filtration phases on pathogen concentration, including week and pathogen as random effects, for each location separately. Ct values for a particular pathogen were included only if the pathogen was detected in samples from all three filtration phases during the same week. Confidence intervals were calculated using the confint command in R, p-value of ≤0.05 was considered significant in all analyses.A two-sided Further information on research design is available in the Supplementary InformationPeer Review FileSupplementary Data 1Supplementary Data 2Reporting Summary