The clinical impact of nucleic acid amplification (NAA) tests on reducing delayed diagnosis and misdiagnosis of pulmonary TB (PTB) has rarely been investigated. PTB patients were classified into a frontline NAA group, an add-on NAA group, and a no NAA group. The outcomes of interest were the proportion of PTB case died before anti-TB treatment, the interval between sputum examination and initiation of treatment, and misdiagnosis of PTB. A total of 2192 PTB patients were enrolled, including 282 with frontline NAA, 717 with add-on NAA, and 1193 with no NAA tests. Patients with NAA tests had a lower death rate before treatment initiation compared to those without NAA tests  in all cases. Patients with frontline NAA compared to those with add-on NAA and those without NAA, had a shorter interval between sputum examination and treatment initiation in all cases (3 days vs. 6 days (p < 0.001), vs 18 days (p < 0.001)), and less misdiagnosis in smear-positive cases (1.8% vs. 5.6% (p = 0.039), vs 6.5% (p = 0.026)). In conclusion, NAA tests help prevent death before treatment initiation. Frontline NAA tests perform better than add-on NAA and no NAA in avoiding treatment delay in all cases, and misdiagnosis of PTB in smear-positive cases. Rapid diagnosis of TB with early initiation of anti-TB treatment is pivotal in the End TB strategy2. Delayed diagnosis of TB in critically ill patients may result in death before initiation of anti-TB treatment, which can be as high as 11% to 24% of the mortality cases in Taiwan4. Traditional microbiological tests for the diagnosis of TB include smear for acid-fast bacilli (AFB) and culture of Mycobacterium tuberculosis (MTB). The long turnaround time of mycobacterial cultures limits its application as a rapid diagnostic tool.Pulmonary tuberculosis (TB) is an airborne transmitted infectious disease associated with high morbidity and mortality. In 2019, there were an estimated 10 million new TB cases and 1.2 million TB deaths globally6. NAA tests are more sensitive than smear microscopy and have shorter turnaround time than mycobacterial cultures7. However, studies on the impact of Xpert test on increased detection of TB cases reported conflicting findings9. In addition, whether implementation of NAA tests may reduce delayed diagnosis and misdiagnosis of TB has rarely been investigated11.Nucleic acid amplification (NAA) tests for TB diagnosis have been commercially available for more than 20 years. Xpert MTB/RIF (Xpert) is a widely used NAA tests for diagnosis of TB and detection of rifampicin resistance. When used as an initial diagnostic test replacing smear microscopy, the sensitivity of Xpert was 98% for smear-positive pulmonary TB and 68% for smear-negative pulmonary TB, with a specificity of 99% for both12, or as an add-on test after smear microscopy14. When used as an initial diagnostic test, NAA tests can facilitate early diagnosis of TB. When used as an add-on test, NAA tests can improve diagnostic accuracy by excluding non-TB disorder, such as non-tuberculous mycobacterium (NTM). The latest Taiwan Guidelines for TB diagnosis and treatment recommend NAA tests as an initial diagnostic test, together with smear microscopy and culture15. However, NAA tests were frequently performed as an add-on test following a positive smear-microscopy result to exclude NTM, or after a negative smear-microscopy result if TB was suspected. TB is a notifiable disease in Taiwan and must be reported to the Taiwan Centers for Disease Control (CDC)16. However, patients may be advised by clinicians to stop anti-TB treatment after treatment initiation if a misdiagnosis is noted and diagnosis is changed to non-TB17. We hypothesized that the use of NAA tests may help to reduce delayed diagnosis, death before initiation of anti-TB treatment and misdiagnosis of TB. The aim of this retrospective cohort study was to evaluate the clinical impact of NAA tests on (1) death before initiation of anti-TB treatment, (2) delayed diagnosis of TB assessed by the interval from sputum examination to treatment initiation, and (3) misdiagnosis of TB.NAA tests have been recommended as an initial diagnostic test replacing smear microscopyOut of 4271 presumptive pulmonary TB patients with sputum NAA tests in 2017 and 2018 in the seven study hospitals, 1084 had been diagnosed with active pulmonary TB and reported to Taiwan CDC. Of the 1084 patients, 66 had missing information  and 19 were younger than 20 years old. These 85 patients were excluded, and the remaining 999 patients were included for analysis. Of those, 282 (28.2%) had frontline NAA, and 717 (71.8%) had add-on NAA tests. There were 1193 adult pulmonary TB patients diagnosed in 2013–2014 in the study hospitals that did not conduct NAA tests; these patients were enrolled as the no NAA cohort for comparison , but also significantly prolonged the interval in smear-positive patients (4 days vs. 3 days), due to the fact that a high proportion of patients had add-on NAA, rather than frontline NAA. These findings suggest that the benefit of NAA tests would be different between smear-positive and smear-negative TB cases.Clinical studies evaluating the clinical impact of NAA tests between smear-positive and smear-negative cohorts are limited. Marks et al. reported that the benefits of NAA tests in reducing respiratory isolation and invasive procedures are most significant in smear-positive/culture-negative cases14. A retrospective cohort study in Italy found that using Xpert as an add-on test to smear microscopy increased the proportion of cases diagnosed by 21%24. When compared to frontline NAA without smear microscopy, add-on NAA tests following smear microscopy were associated with higher TB case detection rate in previous studies26. Analysis of the impact of different NAA test strategies on improving early diagnosis and decreasing misdiagnosis remains limited. In the present study, we found that, when compared to add-on NAA, patients with frontline NAA tests had a shorter interval between sputum examination and treatment initiation, and more patients initiated anti-TB treatment at day 7 and day 14, irrespective of the sputum smear status.There are two different strategies when applying NAA tests for TB diagnosis, either frontline tests as initial diagnostic tool, or add-on tests used when smear microscopy results are available and additional diagnostic tests are necessary17. Houben et al. reported that false-positive TB diagnoses had profound consequences for TB patients and prevention efforts, yet were usually overlooked in policy decision making27. Studies have reported that Xpert tests may reduce empirical treatment and overtreatment of TB30. In the present study, smear-positive patients with frontline NAA tests had fewer cases of misdiagnosis than those with add-on NAA. In this study, a significant proportion of smear-positive patients with add-on NAA initiated anti-TB treatment before the results of NAA tests were available, which may lead to misdiagnosis of TB. Our findings indicated that although both frontline and add-on NAA tests can improve the accuracy of TB diagnosis and accelerate treatment initiation, the clinical benefits of NAA tests are more prominent when used as frontline strategy. In areas with adequate medical resources, NAA tests should be used in conjunction with smear microscopy as initial tests, rather than add-on tests that are performed when the results of smear microscopy are available.Misdiagnosis of TB has rarely been investigated before. Chiang et al. reported that 13.9% of the TB patients notified in Taipei had their TB diagnosis changed and were advised to stop anti-TB treatmentThis study had several limitations. As a retrospective study with large sample size, some relevant clinical characteristics, such as the presence of cavitation, and underlying comorbidities, have not been included. In addition, the sputum smear status were not similar between patients with and without NAA test. However, we have performed separate subgroup analysis in smear-positive and smear-negative cases. Great variation of the proportions of cases with frontline NAA, add-on NAA, and no NAA was noted in each participating hospital, suggesting that application of NAA was highly heterogeneous in different hospitals. Finally, Taiwan is an area with low HIV incidence. The HIV status of enrolled cases was not included in our study design, but the general HIV rates in incident TB cases in Taiwan was only approximately 2.5–3%.In conclusion, this multicentre, retrospective cohort study enrolled more than 2000 pulmonary TB patients and analysed the clinical benefit of NAA tests as frontline tests or add-on tests. We found that inclusion of NAA tests in diagnosis of pulmonary TB can improve diagnosis accuracy in smear-positive cases and accelerate treatment initiation in both smear-positive and smear-negative cases. The clinical benefits are more significant for frontline NAA than for add-on NAA tests. It highlighted the clinical benefits of NAA tests in diagnosis and treatment initiation of pulmonary TB, and the potential superiority of the frontline strategy compared to the add-on strategy. In TB endemic areas with adequate medical resources, inclusion of NAA tests as an initial diagnostic tool should be considered to optimise TB diagnostic accuracy and early treatment initiation.This was a retrospective cohort study performed in seven referral hospitals in Taiwan. Pulmonary presumptive TB patients, who had undergone NAA tests from January 2017 to December 2018 and were subsequently notified to Taiwan CDC with diagnosis of pulmonary TB, were enrolled. Pulmonary TB patients notified from January 2013 to December 2014 without NAA tests were included as a reference group (no NAA group), because NAA test had not yet been recommended as an initial diagnostic test by the Taiwan TB guidelines during that period. Exclusion criteria were: patients below 20 years old, active TB patients without pulmonary involvement, and active TB patients without sputum smear microscopy. Data regarding the age, sex, type of NAA tests, and detailed information of smear microscopy, including examination date and results, were collected from medical chart reviews. Patients with ≥ 1 positive AFB smear were defined as smear-positive cases, and patients never having a positive AFB smear were defined as smear-negative cases. The study protocol was approved by the Institutional Review Boards of all participating hospitals, and informed consent requirement was waived .Among the seven participating hospitals, two used Roche Diagnostics  Cobas Amplicor Mycobacterium tuberculosis assay, three used Xpert MTB/RIF , and two used both Xpert MTB/RIF and in-house TB PCR tests. Frontline NAA was defined as NAA test requested before or concomitantly with smear microscopy; add-on NAA was defined as NAA test requested after obtaining results from smear microscopy.Outcomes of interest in the present study included the number and proportion of TB patients that died without anti-TB treatment, the interval between sputum examination and initiation of anti-TB treatment (delayed diagnosis), and misdiagnosis of TB. Misdiagnosis of pulmonary TB was defined as notified pulmonary TB cases, who were advised by clinicians to stop anti-TB treatment before completion of the treatment course and were de-notified, due to TB misdiagnosis. Information regarding diagnosis change (misdiagnosis), date of death, and treatment initiation were retrieved from the TB registration database of Taiwan CDC. The date of first sputum examination, including smear microscopy, mycobacterium culture, and NAA tests, were acquired from chart reviews.Comparisons of demographic and clinical characteristics were performed using a chi-square test or Fisher’s exact test for categorical variables, and the t test or Mann–Whitney U test for continuous variables, depending on data distribution. Death without anti-TB treatment, misdiagnosis, and delayed diagnosis of TB were compared between groups with different NAA strategies accordingly. Analysis of delayed diagnosis and misdiagnosis included patients who had initiation of anti-TB treatment after sputum examinations; those who died before anti-TB treatment and those who had the initiation of treatment before sputum examination were excluded. Since the impact of NAA tests in diagnosis and treatment of tuberculosis may be different between smear-positive and smear-negative cases, analysis was stratified by smear microscopy results. A Kaplan–Meier analysis was conducted, and log-rank tests were performed to compare the time to treatment initiation among patients with different NAA strategies. Sensitivity analysis was performed in culture-confirmed cohorts to confirm the findings from the overall population. Statistical analyses were performed using SPSS version 20.0 software  and Stata Version 15 .The study protocol was approved by the Institutional Review Boards of all participating hospitals, and informed consent requirement was waived .The research was performed in accordance with the Declaration of Helsinki.Supplementary Tables.


To examine the comparative efficacy and safety of cognitive enhancers by patient characteristics for managing Alzheimer’s dementia (AD).Systematic review and individual patient data (IPD) network meta-analysis (NMA) based on our previously published systematic review and aggregate data NMA.MEDLINE, Embase, Cochrane Methodology Register, CINAHL, AgeLine and Cochrane Central Register of Controlled Trials up to March 2016.80 randomised controlled trials (RCTs) including 21 138 adults with AD, and 12 RCTs with IPD including 6906 patients.Cognitive enhancers  alone or in any combination against other cognitive enhancers or placebo.We requested IPD from authors, sponsors and data sharing platforms. When IPD were not available, we used aggregate data. We appraised study quality with the Cochrane risk-of-bias. We conducted a two-stage random-effects IPD-NMA, and assessed their findings using CINeMA .We included trials assessing cognition with the Mini-Mental State Examination (MMSE), and adverse events.Our IPD-NMA compared nine treatments (including placebo). Donepezil (mean difference (MD)=1.41, 95% CI: 0.51 to 2.32) and donepezil +memantine  improved MMSE score  compared with placebo. According to P-score, oral rivastigmine  and donepezil  had the least favourable safety profile, but none of the estimated treatment effects were sufficiently precise when compared with placebo . For moderate-to-severe impairment, donepezil, memantine and their combination performed best, but for mild-to-moderate impairment donepezil and transdermal rivastigmine ranked best. Adjusting for MMSE baseline differences, oral rivastigmine and galantamine improved MMSE score, whereas when adjusting for comorbidities only oral rivastigmine was effective.The choice among the different cognitive enhancers may depend on patient’s characteristics. The MDs of all cognitive enhancer regimens except for single-agent oral rivastigmine, galantamine and memantine, against placebo were clinically important for cognition (MD larger than 1.40 MMSE points), but results were quite imprecise. However, two-thirds of the published RCTs were associated with high risk of bias for incomplete outcome data, and IPD were only available for 15% of the included RCTs.CRD42015023507. This is one of the most comprehensive systematic reviews and network meta-analysis of cognitive enhancers including individual patient data for Alzheimer’s dementia to produce treatment recommendations by patient characteristics.We followed the methodologically rigorous guidelines in the Cochrane Handbook for systematic reviews, and assessed credibility in the results using the Confidence in Network Meta-Analysis tool.Access to individual patient data allowed us to (1) observe minor differences between the original published results and our reanalysis, potentially due to differences in imputation methods for missing data or because original studies have excluded some patients, and hence have used a smaller sample size, (2) overcome potential reporting bias and (3) assess for potential effect modifiers that were not reported in the original publications  and explore for treatment-by-covariate interactions on the patient-level.Two-thirds of the included randomised controlled trials (RCTs), were associated with high risk of bias for incomplete outcome data due to attrition.We were unable to include individual patient data for all RCTs , highlighting potential retrieval bias.Our literature searches were conducted 5 years ago and additional relevant studies may be available. However, obtaining individual patient data in a timely manner was very challenging and required more time than anticipated. Similar to all systematic reviews, the evidence should be updated regularly.Alzheimer’s dementia (AD) is the most common type of dementia.5In AD, disease severity and sex are potential effect modifiers. However, aggregate data and covariates of interest  are not consistently reported across randomised clinical trials (RCTs).We reported our results according to the Preferred Items for Systematic Reviews and Meta-Analysis (PRISMA) statement for NMA and PRISMA-IPD.7 8The research question and protocol were based on our previous systematic review and NMA.10.1136/bmjopen-2021-053012.supp1Supplementary data10.1136/bmjopen-2021-053012.supp2Supplementary dataWe updated our previous systematic review,We contacted the corresponding author followed by the next-in-order author, as presented in each eligible RCT, to obtain IPD. The author contact process was part of an RCT that our team conducted to assess methods that may optimise response rates for IPD retrieval.We appraised study quality using the Cochrane risk of bias tool.When at least 10 studies were available for each treatment against placebo, publication bias and small-study effects were examined visually using the comparison adjusted funnel plot under the fixed-effect model.15We performed a descriptive analysis using frequencies and distributions of the characteristics of the included patients and treatments. For each outcome, we present the network geometry according to IPD availability. We conducted a two-stage IPD analysis, whereby data were analysed separately in each trial in the first stage and the trial parameter estimates were synthesised in a random-effects meta-analysis or NMA in the second stage.The summary treatment effects are presented using the OR or mean difference (MD) along with their corresponding CIs and prediction intervals (PIs).17 18Not applicable.After screening 20 410 titles and abstracts and 1968 full-text articles, 96 studies fulfilled the eligibility criteria; 80 unique studies and 16 companion reports .Of the 80 unique RCTs, 55 reported at least one industry-sponsored funder . In the remaining studies, nine were publicly-sponsored and 16 did not report any information about funding. We requested IPD by contacting the corresponding authors for 80 RCTs that included 21 138 participants. None of the original authors shared their IPD. Fifteen commercial sponsors were then contacted and 6 (40%) sponsors shared their data through proprietary sponsor-specific platforms. The six sponsors were contacted for 46 RCTs (14 580 participants), and we obtained IPD for 30%  of these RCTs . The study flow for obtaining IPD is depicted in We were able to include 12 (6906 patients) of 14 RCTs in our NMA due to incompleteness of provided IPD . The numMost included studies (33%) were multinational. The mean age of patients ranged from 61 to 86 years. The majority of the RCTs included patients with mild–moderate AD (55%), although the diagnostic criteria used for AD varied widely Using the Cochrane risk-of-bias tool, allocation concealment was at low risk of bias for 43% and blinding of participants and personnel was low for 64% of the RCTs . One-thi2=1.95, and correlation coefficient −0.45 (–0.76 to –0.01) reflecting the belief that the propensity for publication was associated with the observed effect size.All IPD provided were checked for consistency and results from published RCTs were reproduced and provided in 2=4.36, 13 df, p value=0.987; AE: χ2=3.57, 6 df, p value=0.735). Below we present the main analysis results compared with placebo. Additional analyses are presented in In both MMSE and AE outcomes, on average there were no important concerns regarding the transitivity and consistency assumptions  and 11 619 participants. Nine RCTs (3625 patients) contributed IPD and 47 RCTs (7994 patients) contributed aggregated data to the NMA. Two studiesStudies in this NMA compared all available treatments. Donepezil  and donepezil +memantine  were superior to placebo in terms of MMSE score . TransdeStudies in this NMA compared all available treatments. Donepezil improved MMSE score significantly . Assuming an MCID of 1.40, results were in agreement with the NMA of IPD and aggregate data, and donepezil +memantine  was likely the most effective in improving MMSE score (P-score=76%).Studies in this NMA compared placebo, donepezil, oral rivastigmine, transdermal rivastigmine, galantamine and memantine. Donepezil  and transdermal rivastigmine  were superior to placebo, but none of the point estimates reached a previously suggested MCID.Overall, additional analyses using both IPD and aggregate data were in agreement with the findings of the main analysis . CognitiAccounting for the impact of the outlier studies, galantamine +memantine was the second-best cognitive enhancer  after donepezil +memantine . Using only IPD adjusted for comorbidities suggested that oral rivastigmine improves MMSE score . Similarly, using IPD adjusted for cognitive impairment assessed with MMSE at baseline suggested that oral rivastigmine  and galantamine  improve MMSE score, but in a future study, results are only stable for galantamine.2=96%) compared also to the Rhodes et al2=73%), including only patients with moderate-to-severe AD , restricting to industry-sponsored trials  and using IPD only .Heterogeneity in NMA was high , followed by donepezil  and galantamine +memantine , yet in these comparisons the odds of experiencing an AE were imprecise and not importantly different from placebo . ConfideStudies in this NMA compared all available treatments. Results were mainly consistent with NMA of IPD and aggregate data, but memantine was 0.70 times less likely to experience an AE than placebo, with an OR ranging from 0.51 to 0.97 (P-score=77%).Studies in this NMA compared placebo, donepezil, oral rivastigmine, transdermal rivastigmine, galantamine and memantine. Results were on average consistent with NMA of IPD and aggregate data.Additional analyses using both IPD and aggregate data, showed that memantine was 0.61 times less likely to experience an AE than placebo when using study duration as a covariate, with an OR ranging from 0.37 to 0.93 (P-score=88%). Restricting to low risk of bias for incomplete outcome data, galantamine was associated with significantly lower odds of an AE .2=22%) compared with the Turner et al2=0%), low risk of bias for incomplete outcome data , patients with moderate-to-severe cognitive impairment  and when adjusting for study duration (between-study variance=0.03), year of publication (between-study variance=0.02), mean age (between-study variance=0.02) or sex (between-study variance=0.03).Heterogeneity in NMA was low , a larger improvement in cognitive performance was observed for donepezil and memantine, and their combination (donepezil +memantine), and these efficacy-related results are expected to also be reflected when a future study becomes available. The least effective cognitive enhancer in participants with moderate-to-severe cognitive impairment was oral rivastigmine. For patients with mild-to-moderate impairments based on MMSE scores, donepezil and transdermal rivastigmine were most likely the best performing cognitive enhancers. For patients with moderate-to-severe cognitive impairment, cognitive enhancers were well tolerated. For patients with mild-to-moderate cognitive impairment, all except for memantine and its combination with transdermal rivastigmine, were associated with increased odds of an AE, yet none of these results reached statistical significance. Overall, memantine was associated with lower odds of an AE than placebo, yet this was statistically significant only in the subnetwork analysis including aggregate data  and the meta-regression analysis using study duration as a covariate. However, acknowledging for heterogeneity in the network, PIs suggested that results are inconclusive and the odds of AE could not be differentiated between memantine and placebo. Of note, the accuracy of AE reporting may be impacted by the degree of cognitive impairment. Using IPD only and adjusting for MMSE baseline differences,  a study that we previously were unable to include since arm-level data were not reported in the RCT publication,An important finding of our review is that the two-thirds of the published RCTs, were associated with high risk of bias for incomplete outcome data due to attrition, and the majority of these RCTs used the last-observation-carried-forward technique for missing data. This approach may bias results favouring cognitive enhancers, since the dropout rates were greater in the treatment group compared with the placebo group in 63% of the included studies and because dementia is a progressive disease. Of the 27 studies comparing treatment against placebo and reporting the number of dropouts, 17 studies had a greater dropout rate in the treatment group ; placebo group: median dropout rate=21%, IQR (15%–31%)). Last-observation-carried-forward is an inappropriate imputation method for AD studies, since it ignores expected deterioration of the patient’s condition and stabilises the outcome at the value observed at the time of dropout .Our study has limitations worth mentioning. First, we were unable to include IPD for all eligible studies (only 15% of the included RCTs shared their IPD), highlighting potential retrieval bias for IPD. However, recent simulations have shown that combining IPD and aggregate data in an NMA can significantly improve precision, reduce bias and increase information compared with NMA relying on aggregated data alone.32We expect that our findings will increase scientific knowledge, because people with AD require personalised medicine to optimise their healthcare. Well-conducted meta-analyses of IPD are considered the ‘gold-standard’ and influence patient care since patient-level data can be provided to facilitate tailored decision-making. However, results from meta-analyses of IPD are likely subject to retrieval bias and awareness of these limitations and their potential impact on findings is required .


Staphylococcus aureus (22.2%), Pseudomonas aeruginosa (27.8%), and Klebsiellaspp (25.0%). Patients self-medicating with antibiotics had significantly higher rates of multidrug resistance. The multiplex PCR test was more accurate in identifying multidrug resistance and resulted in a quicker initiation of therapeutic antibiotics compared with instances where a bacterial culture was initially performed, with an average of 26.8 h vs. 40.4 h, respectively. The hospital stay was also significantly shorter by an average of 2.5 days when PCR was used as an initial assessment tool for secondary bacterial infections. When adjusted for age, COVID-19 severity, and pulmonary disease, over-the-counter use of antibiotics represented a significant independent risk factor for a prolonged hospitalization (AOR = 1.21). Similar findings were observed for smoking status (AOR = 1.44), bacterial superinfection (AOR = 1.52), performing only a conventional bacterial culture (AOR = 1.17), and a duration of more than 48 h for bacterial sampling from the time of hospital admission (AOR = 1.36). Multiplex PCR may be a very effective method for diagnosing secondary bacterial infections in COVID-19 individuals self-medicating with antibiotics. Utilizing this strategy as an initial screen in COVID-19 patients who exhibit signs of sepsis and clinical deterioration will result in a faster recovery time and a shorter period of hospitalization.The multiplex PCR is a powerful and efficient tool that was widely used during the COVID-19 pandemic to diagnose SARS-CoV-2 infections and that has applications for bacterial identification, as well as determining bacterial resistance to antibiotics. Therefore, this study aimed to determine the usability of multiplex PCR, especially in patients self-medicated with antibiotics, where bacterial cultures often give false-negative results. A cross-sectional study was developed in two COVID-19 units, where 489 eligible patients were included as antibiotic takers and non-antibiotic takers. Antibiotic takers used mostly over-the-counter medication; they suffered significantly more chronic respiratory conditions and were self-medicated most often with cephalosporins (41.4%), macrolide (23.2%), and penicillin (19.7%). The disease severity in these patients was significantly higher than in non-antibiotic takers, and bacterial superinfections were the most common finding in the same group (63.6%). Antibiotic takers had longer hospital and ICU admissions, although the mortality rate was not significantly higher than in non-antibiotic takers. The most common bacteria involved in secondary infections were  Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), was initially identified in late 2019 and quickly developed into a pandemic, as stated on 11 March 2020 by the World Health Organization . The yetStreptococcus pneumoniae, Staphylococcus aureus, Mycoplasma pneumoniae, influenza A, parainfluenza, rhinovirus, enterovirus, respiratory syncytial virus (RSV), and other coronaviruses [Coinfections occur simultaneously with the initial SARS-CoV-2 infection, while superinfections develop during the disease’s clinical course. However, these two entities are often not described explicitly in the literature, resulting in inconsistent reporting of the rates of coinfections and superinfections in COVID-19 patients . COVID-1aviruses ,15. CurrMultiplex polymerase chain reaction (PCR) panels may promptly detect the presence of respiratory infections and may aid in defining antimicrobial indications and medication selection . WheneveAntibiotic resistance is a serious danger since every year resistant bacteria claim at least 700,000 lives worldwide . Antibiop-value = 0.003). Similarly, there were 24 (12.1%) patients taking antibiotics who suffered from chronic obstructive pulmonary disease (COPD), compared with 17 (5.8%) in the group of non-antibiotic takers .Data collection identified 489 eligible patients that were hospitalized for COVID-19 and suffered a secondary bacterial infection. These included 198 individuals that admitted self-medicating with antibiotics since the onset of symptoms. The comparison of the background data from the group of antibiotic takers and non-antibiotic takers in Of the 198 patients with COVID-19 and a secondary bacterial infection, the majority self-medicated with cephalosporins (41.4%), followed by macrolide (23.2%), and penicillin (19.7%), as presented in p-value = 0.040). The proportions of secondary bacterial infections were statistically significantly different, where bacterial superinfections represented the highest proportion in antibiotic takers (63.6%), compared with 52.6% in non-antibiotic takers . The COVID-19 severity and lung involvement were higher in the group of antibiotic takers, where ground glass opacities spread over more than 60% of the pulmonary area were seen in 11.1%, compared with 6.2% in non-antibiotic takers . Additionally, 24 (12.1%) of the antibiotic takers developed a severe SARS-CoV-2 infection, compared with 21 (7.2%) in the non-antibiotic takers group . In the group with the highest number of severe SARS-CoV-2 infections there was a greater need for invasive ventilation , and ICU admissions . The same patients who self-medicated with antibiotics had a longer stay in the ICU  and a higher duration between symptom onset and death . However, overall mortality did not differ significantly between the two main study groups.An in-depth analysis of COVID-19 patient outcomes determined multiple statistically significant differences between patients who self-medicated with antibiotics and non-antibiotic takers . There wp-value = 0.004), respectively . ALT and AST levels were also significantly increased in patients that self-medicated with antibiotics , respectively . Other significant differences in the biological parameters were determined in the study groups’ median values of the inflammatory markers IL-6 , and fibrinogen .The comparison of the biological parameters of the two independent study groups presented in p-value = 0.351).A Kaplan–Meyer curve was plotted in p-value < 0.001). On the contrary, the PCR and bacterial culture methods did not show significant differences in false-negative results in the group of non-antibiotic takers . Sputum samples were positive in 91.7% of specimens analyzed with PCR, and only 51.4% when analyzed via conventional cultures . Similarly, the PCR test was significantly more accurate in blood and urine samples taken from the same 72 antibiotic takers with COVID-19 , and Pseudomonas aeruginosa . In the group of non-antibiotic takers, the only significant difference observed was the proportion of positive tests for Enterococcus faecalis .The most commonly involved pathogen in the group of antibiotic takers was p-value = 0.035). Other significant differences were in macrolide resistance . Additionally, multidrug resistance was more prevalent in the group of 198 antibiotic takers hospitalized with COVID-19 . Lastly, the same group was found with a significantly higher number of infections with more than two pathogens .The multiplex RT-PCR proved its efficiency in determining antibiotic resistance, where significant changes were only observed in the group of antibiotic takers . A totalAs presented in p-value < 0.001), respectively . The duration of time from hospital admission until therapeutic antimicrobial treatment was initiated also differed significantly between both study groups by the test used for bacterial identification , respectively . Lastly, COVID-19 patients self-medicating with antibiotics that had an initial assessment for secondary bacterial infection by multiplex PCR spent 12.5 days in the hospital, compared with 14.9 days spent by those who had an initial test for bacterial identification using a conventional culture method . Non-antibiotic takers that had an initial assessment for secondary bacterial infection by multiplex PCR spent 12.0 days in the hospital, compared with 14.5 days spent by those who had an initial test for bacterial identification using a conventional culture method .A comparison of COVID-19 patient outcomes determined by the status of antibiotic use and the first bacterial identification test performed after hospital admission is presented in p-value = 0.007, vs. non-antibiotic takers log-rank p-value = 0.013).The Kaplan–Meyer tests presented in p-value = 0.042). Similar findings were observed for smoking status , bacterial superinfection , performing only a conventional bacterial culture , and a duration for bacterial sampling longer than 48 h from hospital admission .The risk factor analysis presented in In this study, bacterial superinfections were the most common patient presentation. It was observed that COVID-19 patients self-medicated with antibiotics were more likely to have a superinfection in 63.6% of the hospital admissions for secondary bacterial infections. Among the participants, almost one-third (32.2%) had asked their doctor to prescribe antibiotics for an illness, and this request was significantly related to taking antibiotics for COVID-19. Probably the most common misunderstanding these patients have is that antibiotics can treat the common cold and generally all respiratory infections such as COVID-19. As bacterial cultures often return false-negative results in samples provided by antibiotic takers, the multiplex PCR analysis proved to be a more accurate tool for bacterial identification and a more efficient method of testing, which reduced the duration of hospital stay by an average of 2.5 days.The discrepancy between the results obtained from culture experiments and those derived from a PCR molecular analysis is most likely due to antibiotic use, although the multiplex PCR showed better accuracy even in non-antibiotic takers. PCR targeting the 16S rRNA gene has been shown to effectively detect and identify living or dead bacteria in patients receiving antibiotic treatment and in symptomatic patients with a culture-negative microbiological report ,24. MicrOther investigations show a considerable proportion of culture-negative samples containing bacterial DNA (14%), which is often found using not only a universal pair of primers but also primers targeting particular bacteria. When only individuals with symptomatic illnesses were evaluated, this proportion of culture-negative, PCR-positive samples increased to 27% ,26. ReceStaphylococcus aureus, Klebsiella spp., and Pseudomonas aeruginosa, as the most common bacteria involved in secondary infections in COVID-19 patients. COVID-19 is caused by an extremely contagious infectious pathogen that spreads mostly by droplets and intimate contact. Numerous family infection clusters have been identified, and some of the confirmed cases occurred in hospitals. In a recent study from Belgium, 40 percent of patients had coinfection with Staphylococcus aureus, Haemophilus influenzae and Moraxella catarrhalis as the most often found bacteria with multiple genome copies. In Italy, the frequency of Haemophilus influenzae infections was 56% among COVID-19 patients [Klebsiella spp. and Escherichia coli [Haemophilus influenzae. These differences might be attributed to hospital conditions that vary by region and economy and also by the SARS-CoV-2 strain involved.This study identified patients . Howeverpatients , while ohia coli , rather Antibiotic self-medication is a significant contributor to antimicrobial resistance. One of the study limitations is that only patients hospitalized were assessed, while the number of those self-medicating with antibiotics at home might be much higher. It was previously determined in an Australian study that during the first pandemic wave, 19.5% of the participants used antibiotics to protect themselves against COVID-19 . ParticiThis multicenter research was designed as a cross-sectional study of hospitalized patients with COVID-19 and secondary bacterial infections who self-medicated with antibiotics. The setting comprised two tertiary hospitals in Western Romania, where patients were admitted to the COVID-19 units of the Internal Medicine Department of Timisoara Municipal Emergency Hospital and the Infectious Diseases and Pulmonology Hospital, “Victor Babes” University in the period starting January 2020 until January 2022. The research protocol was approved by the Ethics Committee of the “Victor Babes” University of Medicine and Pharmacy in Timisoara, Romania, and by the Ethics Committee of both hospitals.A search of the database and patients’ paper records was conducted to determine the cases of bacterial coinfection and superinfection in patients with COVID-19 from the two hospitals. Patients who confirmed taking antibiotics at home since the onset of COVID-19 symptoms were included in the “Antibiotic Takers” group, while those who did not take antibiotics before admission were defined as “Non-Antibiotic Takers”. The duration of the self-medicated antibiotic treatment was assumed to be the same as the number of days elapsed from symptom onset until hospital admission, since the recall ability of patients was inconsistent and most of them decided to take antibiotics close to initial symptom onset. A further database and paper records search was necessary to determine the type of bacterial identification test performed initially to diagnose the secondary bacterial infection, either by a multiplex RT-PCR test or a conventional bacterial culture.We used a convenience sampling approach to determine the ideal sample size, which was determined to be at least 385 patients, for a 5% margin of error at a 95% level of confidence. The inclusion criteria were set for all patients over 18 years old with a history of hospital admission for SARS-CoV-2 infection diagnosed by real-time polymerase chain reaction (RT-PCR) in the two hospital departments involved in the current study. All patients were included if their records mentioned a secondary bacterial infection diagnosed during their hospital stay. Patients with immune deficiencies and immune-suppressive therapy were excluded from the study. A secondary bacterial infection was considered to be either a coinfection or a superinfection. Coinfections were defined as those cases diagnosed during the first 48 h of COVID-19 hospitalization, and superinfections as cases diagnosed after 48 h of hospitalization.The variables considered important for evaluation were background data , patient outcomes, including the number of days from symptom onset until hospitalization, COVID-19 complications, type of secondary bacterial infection (coinfection/superinfection), tests performed for bacterial identification , pulmonary ground-glass opacities, COVID-19 severity, oxygen supplementation , ICU admission, duration of ICU admission, the number of days elapsed between first COVID-19 symptoms until death, mortality, and duration of hospital stay. Biological parameters that were assessed included the complete blood count , kidney function tests , liver function tests , and inflammatory markers . For microbial identification, we considered the number of positive specimens were counted , the pathogens involved, the percentage of antibiotic resistance given by PCR or culture tests, the percentage of multidrug resistance, the number of pathogens identified, time of sampling (before or after 48 h since admission), numbers of specimens taken, the time elapsed from sampling until results were given, the time elapsed from hospital admission until therapeutic antibiotic initiation, and lastly, the percentage of discontinued, changed, or continued antibiotics.The fluid and solid samples were taken from COVID-19 patients for analysis according to standard microbiological methods at the Microbiology Unit of the two clinics involved in the study. The standard microbiological method included microscopic observation of the sample after Gram staining and the inoculation of the sample in growing media . The conventional plate culture is a qualitative and quantitative technique that relies on the sample being deposited in or on an agar layer in a Petri dish. Individual organisms or small groups of organisms occupy a defined spot in the agar and develop to form discrete colonies that are visually counted during incubation. Numerous varieties of agar medium may be used in this manner to grow and count various bacteria, as well as to test for antibiotic susceptibility ,35. The t-test . A Kaplan–Meyer curve was plotted for the probabilities of the duration of hospital stay by the type of bacterial identification test employed. Lastly, a multivariate analysis adjusted for confounding factors was performed to determine the independent risk factors for prolonged hospital stay in COVID-19 patients with secondary bacterial infections who self-medicated with antibiotics. The significance threshold was set for an alpha value of 0.05.The statistical analysis was performed with IBM SPSS v.26 and MedCalc v.20. The absolute and relative frequencies of categorical variables were computed. For a comparison of the proportions, the Chi-square and Fisher’s tests were employed, while for the comparison of group differences in nonparametric data, the Mann–Whitney test was used. Parametric continuous variables that followed a normal distribution were compared by mean and standard deviation with the Student’s The multiplex PCR tool can be a very efficient diagnostic tool for secondary bacterial infections in COVID-19 patients who self-medicate with antibiotics. Using this method as an initial screening in COVID-19 patients that show signs of sepsis and clinical deterioration will help to shorten recovery time and hospital admission. We have come to appreciate the critical nature of prudent antibiotic usage, particularly during the COVID period, to avoid unneeded antibiotic treatments and halt the emergence of antimicrobial resistance. Additionally, we propose shortening the turnaround time for tests in the diagnostic route for COVID-19 patients in order to reduce the need to take antibiotics, and we argue for the inclusion of antimicrobial stewardship initiatives in the pandemic response.


The purpose of our study is to quantify the extent to which Virchow-Robin spaces (VRS) detected on in vivo MRI are reproducible by post-mortem MRI.Double Echo Steady State 3T MRIs were acquired post-mortem in 49 double- and 32 single-hemispheric formalin-fixed brain sections from 12 patients, who underwent conventional diagnostic 1.5 or 3T MRI in median 22 days prior to death (25% to 75%: 12 to 134 days). The overlap of in vivo and post-mortem VRS segmentations was determined accounting for potential confounding factors.The reproducibility of VRS found on in vivo MRI by post-mortem MRI, in the supratentorial white matter was in median 80% (25% to 75%: 60 to 100). A lower reproducibility was present in the basal ganglia, with a median of 47% (25% to 75%: 30 to 50).VRS segmentations were histologically confirmed in one double hemispheric section.Overall, the majority of VRS found on in vivo MRI was stable throughout death and formalin fixation, emphasizing the translational potential of post-mortem VRS studies. Virchow-Robin spaces (VRS) are the anatomical spaces between the basement membrane of the perivascular astrocytic feet processes (glia limitans perivascularis) and the basement membranes of the blood vessel endothelia.Within the VRS, CSF flows from the subarachnoid space into the brain parenchyma, allowing for exchange with brain interstitial fluid (ISF). Additionally, VRS are involved in the intramural, perivascular connective tissue drainage pathway, from the brain, towards the subarachnoid space.They are thus involved in central nervous system (CNS) debris removal and lymphatic immune gateways. However, large Virchow-Robin spaces (VRS) are a frequent imaging finding in routine clinical MRI scans of the brain. Enlargement of the perivascular spaces has been suggested as a consequence of debris accumulation, e.g., in the course of mucopolysaccharidoses or during collagen deposition in atherosclerosis, but no singular mechanism has emerged as yet. In multiple sclerosis, a disease with chronic inflammatory and neurodegenerative components, inflammation, in line with community-based studies of ageing, as well as dilatation associated with chronic brain parenchymal atrophy, were associated with enlarged VRS and perivascular collagen deposition.In healthy control subjects, the size of VRS is below the resolution of current conventional brain MRI scans at 3T. stroke recurrence, vascular-related cognitive impairment, oral anticoagulation-related symptomatic cerebral hemorrhage, and progression of disability in Parkinson’s disease, as well as both present and future cognitive impairment.2023Large VRS are associated with an increased risk for lacunar stroke,in vivo MRI has remained largely unspecified, as it is still unclear at which rate large VRS were preserved after death and post-mortem tissue processing.Detailed histological and molecular analysis of large VRS on post-mortem tissue could further guide our understanding of CNS waste clearance and the influence of vascular pathology on CNS disorders. However, the actual histopathological correlates of VRS seen on The purpose of our study therefore was to quantify the extent to which VRS detected on in vivo MRI are reproducible by post-mortem MRI in 12 individuals who underwent MRI at a median of 22 days before their death and in whom the brain was available for post-mortem processing.Individuals were recruited based on the following criteria: brain MRI within <one year before death; in vivo MRI with sufficient image quality to visualize and segment the VRS; and availability of post-mortem brain tissue from the same patient at our local neuropathology department. This inclusion criterion was met by twelve consecutive patients. Clinical details, including the number of analysed tissue blocks per patient, main clinical and neuropathological diagnoses, the cause of death, the time intervals from in vivo MRI to death, and from death to post-mortem MRI, are summarized in The tissue slicing was performed in the course of neuropathological clinical routine at standardized section levels, including coronal sections at the frontal poles, at the level of the temporal poles, a mid-thalamic section level and occipital sectioning. The availability of these tissue sections for this study was determined by prior neuropathological diagnostic work-up, regions of clinical interest, typically those including lesions, were utilized and not available for our subsequent analysis. The entire available brain tissue was analyzed in each patient. In total we investigated 81 tissue-blocks, of which 49 were double- and 32 single-hemispheric/lobar formalin-fixed brain sections; On a patient basis in median 4 double-hemispheric sections (25%–75%: 3–5), and 2 single-hemispheric sections were included (25%–75%: 0–5) In vivo MRI acquisition was performed in the course of the clinical routine, with imaging adapted to the clinical question and thus not standardized across all patients. However, the imaging protocols were quite similar, as all patients were imaged in our department. A 1.5 Tesla Philips Ingenia MRI with an eight-channel head coil was used in 10/12 subjects. The remaining two subjects were scanned with a Philips Achieva  and a Siemens Vida , respectively. 3D-T1-weighted images were available in all cases. A detailed list of the analyzed in vivo MRI sequences is available in Supplementary Table 1.From the included 12 subjects, a total of 49 double- and 32 single-hemispheric/lobar formalin-fixed brain sections, with a slice thickness of around 1 cm were obtained for the post-mortem MRI analysis, the sections were cut prior to formalin fixation. Tissue sections were axially oriented in one subject (#22-19), which was anaylsed axially, wherease coronal reformates were used in all the other cases. Post-mortem scanning was performed with a Siemens Vida 3T scanner.Formalin-fixed tissue blocks were individually placed in a flat plastic container and fully emerged in Flourinert.Double Echo Steady State (DESS) images were acquired using the built-in spine coil of the table, which has 72 channels in 12 segments, of which we used four, and the Siemens “multiflex surface coil,” with 18 channels on top of the specimen.T2-weighted DESS images were acquired with an echo time of 5.6 ms, a repetition time of 17 ms, and a flip angle to 25°. The isotropic resolution was 0.4 mm (matrix: 468 × 576), and the acquisition time, per tissue block, was around 30 to 50 min. Due to the immersion in Flourinert the brain is surrounded by extremely low signal intensity despite the T2-weighted contrast. Histological images were manually co-registered to the aligned post-mortem and in-vivo MRI images.To test if VRS segmented on MRI correspond to VRS histologically, proof of concept was tested in one double hemispheric section from ID #120-19. The section was stained with Hematoxylin Eosin, Elastica van Gieson, and Luxol fast-blue myelin stain as reported previously. on the in vivo data set  and subsequently on the post-mortem set  in the entire brain. Lesions were masked from the analysis (LH). The in vivo segmentation were performed without knowledge of the post-mortem images. As suggested previously (STRIVE-criteria), VRS were defined as CSF iso-intense lines following the course of vessels in the supratentorial white matter and deep gray matter.In a first step, VRS were manually segmented with 3D-Slicerin vivo MRIs based on anatomical landmarks with Freeview/FreeSurfer. VRS segmentations were not used as additional information/landmarks for the registration process.The post-mortem MRIs were manually and rigidly co-registered with the corresponding in vivo and the post-mortem VRS segmentations were manually quantified for each tissue-block using Freeview/FreeSurfer by LH/SM. To account for the inherent tissue distortion that occurs between in vivo and post-mortem imaging and the limited slice thickness (∼1cm), the analysis was limited to one section level within each tissue-block with the highest spatial correspondence of anatomical landmarks. VRS were counted on in vivo MRI scans with both segmentations from in vivo and post-mortem MRI overlaid at 50% opacity in Freeview. We collected the counts of VRS where the segmentations overlapped or did not overlap for each tissue block  and supratentorial white matter. The rates are provided with medians and a 25% to 75% range.The ratio of in vivo or post-mortem MRI, which was the case in 4/20 blocks with deep gray matter, we did not calculate or assign a detection rate.In tissue blocks with no VRS on either in vivo MRI-to-death interval [days], the death-to-post-mortem MRI interval [days], whether tissue-blocks were single-hemispheric/lobar or double-hemispheric, and the surplus of VRS found on post-mortem MRI relative to the total number of VRS in this tissue block, ie., [(post-mortem – in vivo)/(post-mortem + in vivo)] and the total number of tissue blocks analyzed per patient . These models used the individual tissue block as the unit of analysis. To account for the nested structure of the data, tissue blocks were nested within patients. We report the beta for each predictor with its 95% confidence interval and p-value.To account for the potential effect of confounders on the post-mortem detection rate of VRS as imaged on in vivo MRI, we used linear mixed-effect regression models. These models were separately calculated for supratentorial white matter and deep gray matter. Fixed-effect variables included in these two models were the Normality was visually assessed with quantile-quantile- and density plots and qualitatively with the Shapiro-Wilk test.Inter-rater agreement was assessed with inter-class correlation coefficients, which are provided with their respective 95% confidence interval and p-value.Anonymized data, not published in the article, will be shared upon reasonable request from a qualified investigator after approval from the local Ethics committee.The study was approved by the ethics committee of the Medical University Vienna and performed in accordance with the Helsinki Declaration. Written, informed consent was obtained from all subjects or their legal representatives as far as required by Austrian law.Twelve subjects contributed a total of 49 double- and 32 single-hemispheric/lobar formalin-fixed brain sections. The median age at death was 62 years (25% to 75%: 50 to 77-years). The time from MRI to death was a median of 22 days (25% to 75%: 12 to 134 days). The formalin fixation time at post-mortem MRI acquisition was a median of 236 days (25% to 75%: 136 to 407 days). Subjects  died during the course of various conditions, including malignant/metastatic disease (n = 8), decompensated hepatic cirrhosis with cardio-pulmonary resuscitation (n = 1), cerebral vascular occlusion (n = 1), constrictive pericarditis and cerebral tuberculosis (n = 1), and amyloid angiopathy (n = 1)  and thus in all subjects.in vivo MRI were reproduced at post-mortem imaging in the supratentorial white matter, which was in absolute numbers a median of 15 VRS/patient (25% to 75%: 9 to 36).On a patient basis, a median of 80% (25% to 75%: 60 to 100) of VRS found on The deep gray matter was present in 20/81 tissue blocks and absent in three subjects.in vivo VRS reproduced on post-mortem images was lower in the deep gray matter than in the white matter, with a median of 47% (25% to 75%: 30 to 50), and a median of 1.5 VRS/patient (25% to 75%: 0 to 5.8)  .in vivo - post-mortem VRS consistency, nested linear mixed-effect regression models were computed separately for white matter and deep gray matter , there were no effects of the time-interval from in vivo - post-mortem VRS consistency rate.Similarly, in both locations, the surplus of VRS found on post-mortem images, relative to the total number of VRS in this block, was not related to the in vivo - post-mortem VRS consistency than deep gray matter regions from double-hemispheric blocks   .in vivo - post-mortem VRS consistency .The total number of tissue-blocks analyzed per patient was not related to the The inter-rater agreement on VRS within in-vivo MRIs was 0.91  and in the post-mortem MRIs: 0.92 .in vivo – post-mortem VRS reproducibility was 0% (in vivo VRS were present (in vivo VRS count = 1–2). The post-mortem MRI of these blocks showed similarly low VRS counts .In three tissue blocks, the y was 0% . Three oA histological proof-of-concept was achieved in one double hemispheric section from subject #120-19, .in vivo MRI and post-mortem MRI  are increasingly recognized to be involved in the pathogenesis of various neurological conditions. While VRS are readily visualized on in vivo imaging and post-mortem analysis is a necessary prerequisite for further investigation of the histological and biological basis of pathologies that originate in or involve this specific brain compartment. In the present cohort we therefore tested the hypothesis if VRS detected on in vivo MRI are reproduced by post-mortem MRI. In 12 subjects with 81 tissue blocks, the majority of VRS detected on in vivo MRI were reproducible at post-mortem imaging, providing the first quantification of the translational potential of post-mortem VRS studies.Comparability between 16 hemorrhage, and cognitive impairment.1929 Further understanding of the histological and biological processes that occur within large perivascular spaces could thus facilitate our understanding of relevant disease processes. Their enlargement on in vivo MRI has been suggested to occur as a consequence of debris accumulation,29 but VRS are also considered to be fluid-filled spaces and perivascular collagen depositions have been reported in the perivascular space.14 While the histological validation in our study was limited to 1/12 cases, we found that perivascular collagen depositions were associated with VRS that remained visible from in vivo imaging to post-mortem imaging.Large VRS have been associated, among other conditions, with stroke, 80% of VRS seen on in vivo MRI were also observed on post-mortem images. This is a high detection rate considering the underlying variability (see Limitations section). In the deep gray matter, the rate of the VRS seen on in vivo MRI post-mortem was 47%. The lower reproducibility in the deep gray matter could be due to the smaller sample size of this structure and inherent technical reasons, e.g., the registration technique, manual alignment of anatomical landmarks, could be more efficient with gyri and sulci than with deep gray matter structures, and thus, favor alignment of supratentorial white matter. The lower reproducibility of deep gray matter VRS could also reflect biological differences in the VRS. Histologically, VRS in the deep gray matter are distinct from those in the cortex/white matter, as they are surrounded by two distinct layers of leptomeninges separated by a perivascular space that is continuous with the subarachnoid space. They are also clinically distinct, as large VRS located around the perforating lenticulostriate arteries are frequently observed due to their developmental origin, associated with glial tunnels during axonal growth of the forming anterior commissure.Within the supratentorial white matter, where VRS are most abundant,in vivo and post-mortem, the interval from in vivo MRI-to-death was short (a median of 22 days), and, although post-mortem images were carefully aligned with in vivo MRI  with its inherent statistical limitations. In order to maximize the available tissue, we thus included all sections available, leading to differences in the total number of analysed tissue-sections across subjects. In median 4 double-hemispheric sections (25%–75%: 3–5) and 2 single-hemispheric sections were included (25%–75%: 0–5). However, the total number of sections analysed per patient did not show significant effects on the The biological processes that lead to the enlargement of VRS will likely differ, e.g., active perivascular inflammation vs. atrophy vs. perivascular collagenosis, etc., and we can not account for this heterogeneity in this cohort.in vivo (in vivo) and overall inter-rater agreement was high (>90% both in vivo and post-mortem). However, we can neither exclude artificial “VRS opening” in the course of tissue fixation or ascertain the rate of VRS that may have evolved between in vivo MRI and death. While the scanners were theoretically available to scan the blocks on the same machine on which the in vivo images were acquired, the nature of formalin tissue fixation does not allow acquisition of the identical sequences used in vivo and thus hinder a 1:1 comparison.We observed higher counts of VRS post-mortem than in vivo  and 3, wThe proof of concept, that the VRS segmented on MRI correspond to enlarged perivascular spaces histologically was performed in on patient (#120-19), in vivo post-mortem detection rate via “false-positive VRS.”, however this theory is not supported by our data, since, on a block basis, the total number of VRS, neither in vivo or post-mortem, were not predictive for the in vivo post-mortem VRS detection rate.The high number of VRS on post-mortem scans could arguably also increase the In conclusion, the majority of VRS found on in vivo MRI, was observed on post-mortem imaging, thus suggesting that histological and biochemical analysis of VRS detected by post-mortem MRI could guide future research in this evolving field.Click here for additional data file.in vivo MRI by Lukas Haider, Simon Hametner, Verena Endmayr, Stephanie Mangesius, Andrea Eppensteiner, Josa M Frischer, Juan Eugenio Iglesias, Frederik Barkhof and Gregor Kasprian in Journal of Cerebral Blood Flow & MetabolismSupplemental material, sj-xlsx-1-jcb-10.1177_0271678X211067455 for Post-mortem correlates of Virchow-Robin spaces detected on


Participants completed self-reported measures of moderate-to-vigorous PA (MVPA), participation in organized activities, and variables derived from the M-PAC framework. Mixed effects models were computed to examine longitudinal changes in MVPA and cognitions as well as whether cognitions prior to COVID-19 predict MVPA during COVID-19. A generalized estimating equations model was computed to examine longitudinal changes for participation in organized activities. Findings indicated that MVPA  and participation in organized activities  significantly decreased during the COVID-19 pandemic. Correspondingly, there were significant decreases in intentions (B = −0.34), identity (B = −0.19), and habit (B = −0.20), though there were increases in behavioral regulation (B = 0.18). No significant changes were found in affective attitudes, instrumental attitudes, perceived opportunity, and perceived capability. Among the baseline M-PAC cognitions, habit (B = 46.28) was the lone significant predictor of MVPA during COVID-19. Overall results suggest that adolescents' PA behaviors and cognitions were negatively impacted by the COVID-19 pandemic, along with promising evidence that restrictions prompted adaptive utilization of behavioral regulation skills. Moreover, habit appears to play a salient role in predicting PA behaviors during uncertain times involving major life disruptions. These findings highlight the need to dedicate additional attention to PA promotion as COVID-19 moves toward an endemic phase, and that interventions targeting habit formation may be critical for helping adolescents better sustain healthy active lifestyles during major life changes.Despite accumulating evidence that has found significant negative declines in physical activity (PA) as a result of the COVID-19 pandemic, little work has sought to understand how PA cognitions have changed during this period and in relation to behavior change during the pandemic. The purpose of the current study was to investigate the changes in adolescents' PA behaviors and cognitions associated with COVID-19 and prospective predictors of PA using the Multi-Process Action Control (M-PAC) framework. Adolescents were recruited from a large school board and a total of 588 participants (M Due to the alarming levels of viral transmission, severity, and inaction, the coronavirus disease 2019 (COVID-19) was declared a global pandemic in March 2020  opportunities  framework Rhodes,  was receThe M-PAC framework has been employed to describe, explain, and predict PA as well as develop interventions to promote PA  to utilize the M-PAC framework to investigate changes in adolescents' MVPA behaviors and PA-related cognitions during the second wave of the COVID-19 pandemic in Ontario, Canada and (2) to investigate whether PA-related cognitions prior to COVID-19 predicted MVPA during the pandemic. Based on prior research showing PA declined among children and youth during the pandemic . Baseline data were collected from grade 11 students across a large school board (consisting of seven secondary schools) in Ontario, Canada. Participation was voluntary and informed consent was obtained from both students and parents prior to data collection. Students willing to participate completed a 20-min online questionnaire that included questions related to lifestyle behaviors, PA cognitions, and wellbeing during class time in Fall 2019—prior to the pandemic. A follow-up of these students occurred ~12 months later in Fall 2020—during which pandemic-related restrictions were in place. Participants were compensated with a $10 gift card for their participation at each time point. All protocols for the ADAPT study were approved by the institutional research ethics board and district school board. Further details regarding recruitment strategy and research design are reported elsewhere  also completed the follow-up questionnaire (retention rate = 47.5%) and this subsample was used for the present study. The overall sample included a slightly greater proportion of females  than males  and slightly more than half the sample identified as White/Caucasian (54%). Complete details of demographic information are shown in From the 2,412 grade 11 students enrolled in the seven secondary schools within the school board, 1,585 agreed and provided consent to participate (66% response rate). Of these students, 146 respondents (9%) completed <5% of the survey and their participation was considered withdrawn, while 186 respondents (12%) did not have the required parental consent for their participation. Further, 15 respondents (1%) were missing data for all variables of interest used in our analyses. Among the baseline sample of 1,238 participants, 588  and ethnicity (White/Caucasian or non-White/non-Caucasian) were dummy coded, with males and White/Caucasian as the reference group.MVPA was measured using the International Physical Activity Questionnaire-Short Form  was assessed by asking participants if they are presently enrolled in organized activities .Behavioral intention for engaging in MVPA was assessed using a single item measure  for the analyses involving PA cognitions. Statistical significance was set at p < 0.05 for all other analyses. All analyses were performed using IBM SPSS version 26.For the longitudinal analysis of participation in organized activities, a generalized estimating equations model was used. Generalized estimating equations is a technique used for modeling repeated binary observations within individuals, and our model was adjusted for gender and ethnicity. Separate mixed effects models with maximum likelihood were computed to measure change within individuals over time in MVPA and across each PA cognition, accounting for correlation of measures within participants (repeated measures) and within schools. All models included random intercepts at the participant and school levels, as well as a random slope for time. Each model was adjusted for gender and ethnicity. Mixed effects models with PA cognitions as the outcome variable were also adjusted for baseline MVPA. To examine whether M-PAC cognitions at baseline predicted MVPA at follow-up, a 2-step mixed effects model was computed with the potential covariates: gender, ethnicity, and baseline MVPA included on Step 1, and baseline M-PAC cognitions entered on Step 2. Given that separate mixed effects models were computed for each of the eight PA cognitions identified in the M-PAC framework, a Bonferroni correction was applied to limit the Type I error rate. Therefore, statistical significance was set at The descriptive statistics for PA behaviors and PA cognitions at the baseline and follow-up assessments are presented in p < 0.001), indicating that participants were 67% less likely to participate in organized activities during the pandemic compared to before the pandemic. Descriptively, among participants who completed the survey at both time points, 59% of participants (n = 346) reported engaging in organized activities prior to the pandemic, while only 33% of participants (n = 191) indicated that they engaged in organized activities during the pandemic as seen in p = 0.004), indicating a reduction of 56.41 min of MVPA during the pandemic compared to before the pandemic.Results from the generalized estimating equations model showed a significant negative effect of time  significantly decreased over time. Affective attitudes , instrumental attitudes , perceived opportunity , and perceived capability  did not significantly change. For regulatory processes, behavioral regulation  was found to have significantly increased over time. Similarly, both reflexive process measures of identity  and habit  were significant, decreasing between baseline and follow-up. These findings indicate that adolescents reported lower PA intentions, identity, and habit as well as greater behavioral regulation during the pandemic compared to before the pandemic.Results from our mixed effects models examining changes in PA cognitions found several significant changes over time. Among the reflective processes, PA intention . This finding indicates that adolescents who reported stronger PA habits prior to the COVID-19 pandemic engaged in greater amounts of MVPA during the pandemic. No reflective or regulatory measures were found to be significant predictors.Complete results of the mixed effects model predicting MVPA during COVID-19 are presented in The present study assessed changes in Canadian adolescents' PA behaviors from pre- to mid-pandemic  and represents the first to investigate changes in PA-related cognitions during this timeframe. Our findings showed that adolescents spent less time engaging in MVPA, while also being less likely to engage in organized activities during the COVID-19 pandemic compared to pre-pandemic levels. In addition, we found that there were significant declines in intentions, identity, and habit as well as increases in behavioral regulation. Importantly, findings indicate that pre-pandemic PA habits were significant in predicting MVPA during the pandemic.Overall, our results suggest that pandemic-related restrictions negatively impacted adolescents' PA behaviors during the first year of the COVID-19 pandemic, which is largely consistent with previous studies  from the Social Sciences and Humanities Research Council of Canada.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Appendiceal orifice inflammation (AOI) or peri-appendiceal red patch is a skip lesion with segments of continuous colitis from the rectum. Frequently observed in ulcerative colitis (UC) patients, this lesion might be associated with proximal extension in some studies. However, the clinical significance of this lesion and long-term outcomes including therapy remain unclear. Thus, the aim of this study was to evaluate the clinical implication of AOI during long-term periods in patients with UC.We retrospectively reviewed 376 patients with UC who performed complete colonoscopic examinations between April 2000 and December 2020. We compared clinical characteristics and outcomes of patients manifesting AOI with those who did not show AOI during a mean follow-up period of 66.1 months. Long-term outcomes included maximal extent of colitis, proximal extension, therapeutic medical histories, UC-related hospitalization, and relapse.Ninety-eight (26.1%) patients showed AOI without evidence of inflammation in the right colon. Mild disease activity at the diagnosis of UC was more included in patients with AOI than in those without AOI. Other baseline characteristics including disease extent, smoking history, external intestinal manifestation, and terminal ileal ulceration were not significantly different between the two groups. During follow-up periods, patients with and without AOI showed no significant difference in proximal extension, Mayo endoscopic score at the last endoscopic examination, UC-related hospitalization, or relapse. Of medication history, patients with AOI were less included in the group treated with high-dose aminosalicylates than those without AOI. However, therapeutic histories of steroids, immunosuppressive agents, and biologics were not significantly different. Of 62 patients with AOI who underwent follow-up colonoscopy, 36 (58.1%) showed resolution of AOI. Clinical outcomes of the resolution group were not different than those of the non-resolution group. Biopsy results of 77 patients with AOI showed chronic active or erosive colitis.Long-term outcomes of UC patients with AOI were not different from those without AOI. Outcomes of resolution and non-resolution groups of AOI patients were not different either. Thus, AOI might have no prognostic implication in distal UC patients. Appendiceal orifice inflammation (AOI) is referred to as peri-appendiceal red patches presented on mucosal erythema, granularity, erosion, and friability by colonoscopy . Since uAOI is more frequently observed in UC than in other colitis. It could lead to an effective diagnosis of UC when it is combined with proctitis . The repThus, the aim of this study was to investigate the frequency of AOI in UC patients with distal inflammatory involvement and determine the clinical significance and long-term prognosis of UC patients with AOI compared with those without AOI.Medical records of patients diagnosed with UC at the Hallym University Sacred Heart Hospital in Anyang, Korea between April 2000 and December 2020 were analyzed retrospectively. The clinical significance and natural course of discrete terminal ileal ulcers in UC patients using these data have been published . The preWe evaluated the following outcomes during follow-up periods. Maximal disease extent was defined as maximum disease extent according to the above disease extent. Proximal extension was defined as proximal progression of colonic inflammation . UC-related hospitalization was defined as having histories of admission at our hospital for UC aggravation or adverse events of medication. Relapse was defined by the use of corticosteroids, azathioprine, 6-MP, or anti-TNF agents followed by clinical or endoscopic aggravation of mucosal inflammation. Colonoscopic examinations were performed by staff and fellows at the department of gastroenterology of Hallym University Sacred Heart Hospital. We evaluated whether AOI remained or resolved through follow-up colonoscopies. Terminal ileal ulcers were defined as definite mucosal breaks on terminal ileum according to our previous study . HistoloP value < 0.05. Continuous variables were compared by Mann-Whitney U test and categorical variables were compared with Chi-square test or two-tailed Fisher’s exact test. History of treatment with azathioprine/6-MP and anti-TNF agents, UC-related hospitalization, and relapse were calculated using the Kaplan–Meier method, and subgroups were compared by the log rank test. The Statistical Package for the Social Sciences (SPSS) version 25.0  was used for all statistical analyses.Clinical manifestations and outcome parameters were compared between patients with AOI and those without AOI. Statistical significance was considered at vs. 54.3%, P = 0.012) (Table P = 0.288). Among 10 patients who discontinued 5-ASA due to adverse events , 2 patients were in AOI positive group, and the resting 8 patients were in AOI negative group (P = 1.000).Of the 376 UC patients, 98 26.1%) had AOI in colonoscopic examination. Figure % had AOIP = 0.285). Maximal extent, proximal extension, and Mayo endoscopic score at the last endoscopy during follow-up periods were not significantly different between the two groups either (Table P = 0.184). The administration of a high dose 5-ASA was lower in the AOI positive group than in the AOI negative group . However, use of medications including corticosteroids, azathioprine/6-MP, and anti-TNF agents was not significantly different between the two groups. A total proctocolectomy was performed for two patients in the AOI negative group. UC-related hospitalization and relapse during follow-up periods were not significantly different between the two groups . In the analysis of the clinical outcomes relative to time, there were no significant differences between both groups in the history of treatment with azathioprine/6-MP and anti-TNF agents, UC-related hospitalization, and relapse  showed resolution of AOI during the follow-up colonoscopy. The median follow-up duration of these patients was 83 months  and the median number of follow-up colonoscopies was 4 times . The median follow-up duration of 26 patients with continuing AOI was 84 months  and the median number of follow-up colonoscopies was 4 times . We compared clinical features between the AOI resolution group and the AOI continuing group. Baseline characteristics including disease activity and extent were not significantly different. Clinical outcomes during follow-up periods including maximal extent, proximal extension, Mayo endoscopic score at the last endoscopy, medication histories, UC-related hospitalization, and relapse were not significantly different between the two groups either (Table Seventy-seven (78.6%) patients had undergone biopsies of AOI lesions. All biopsies were pathologically diagnosed with chronic active or erosive colitis with or without lymphoid hyperplasia. Twenty-two (28.6%) patients were additionally diagnosed with cryptitis, crypt distortion, and crypt abscess as histologic findings indicating active UC.In this study, we found that long-term outcomes including proximal extension, histories of medications, UC-related hospitalization, and relapse of UC patients with AOI were not significantly different from those without AOI. Long-term outcomes were not significantly different between the group with AOI resolved and the group with AOI remained. Clinical implications of AOI in UC patients with distally involved colitis did not show a significance in the natural course.Patchy inflammation endoscopically observed on appendiceal orifice in distal UC patients was prospectively evaluated in 1997 . Since tThe prevalence of AOI in UC varies between studies, ranging from 7.9–75% because of different diagnostic criteria used. A few studies have evaluated the prevalence of AOI in examinations performed regardless of treatment history or disease duration , 12 whilP = 0.122). In addition, maximal extent, histories of medication, UC-related hospitalization, and relapse were not significantly different between the two groups diagnosed with proctitis at the diagnosis of UC. Our study included larger number of patients with longer follow-up periods than previous studies.The significance of AOI also varies among studies reporting different prognoses, disease severity, and risk of proximal extension. Strisciuglio et al. have shown that periappendiceal inflammation is associated with a major extent of UC inflammation and that pediatric UC patients with AOI have a higher grade of inflammation at the ascending colon than those without AOI . Anzai eOur study correlates with other studies in assessing the clinical course. Byeon et al. have reported that AOI has no prognostic implications in terms of remission, relapse, or proximal extension in 94 patients with distal UC . Naves eA randomized, double-blind, controlled trial (ASCEND II) showed that patients treated with a high-dose of 5-ASA (4.8 g/dL) achieved significantly more overall improvement after induction therapy of moderately active UC compared with those with standard-dose 5-ASA (2.4 g/dL) . In a meet al. reported a case that biopsy results in the cecum could discriminate AOI from mucosa-associated lymphoid tissue (MALT) lymphoma in UC [Histologic evaluation of AOI needs to be performed at the first detection. Iwamuro ma in UC . In addima in UC , 14. OurThe current study was conducted in a retrospective and non-randomized manner at a single center. Biases might have occurred due to unrecognized or unmeasured factors. In addition, patients with extensive colitis at the diagnosis of UC were included, although mucosal inflammation did not reach the ascending colon. The possibility of patchy improvement of inflammation secondary to medical therapy could not be ruled out. Our study had included more patients with mild or inactive disease activity in the AOI positive group. It might lead to less use of high-dose 5-ASA or anti-TNF agents in the AOI positive group than in the AOI negative group. Patients who performed colectomy were also not included in the AOI positive group. Lastly, the history of taking aspirin or non-steroidal anti-inflammatory drug (NSAIDs) and information on bowel preparation were not evaluated. NSAID or preparation agent could contribute to inflammation of the right-sided colon.In conclusion, patients with AOI at the diagnosis of UC did not show any differences in long-term outcomes such as progression of disease extent, hospitalization, disease relapse, or medication histories compared with patients without AOI. Lesions were resolved in more than half of patients with AOI during follow-up periods. A further study enrolling a large number of patients is needed to support results of this study.


Background: Guidelines of cardiopulmonary resuscitation (CPR) recommend the use of personal protective equipment (PPE) during the resuscitation of COVID-19 patients. Data on the effects of PPE on rescuers’ stress level and quality of CPR are sparse and conflicting. This trial investigated the effects of PPE on team performance in simulated cardiac arrests. Methods: During the pandemic period, 198 teams (689 participants) performed CPR with PPE in simulated cardiac arrests (PPE group) and were compared with 423 (1451 participants) performing in identical scenarios in the pre-pandemic period (control group). Video recordings were used for data analysis. The primary endpoint was hands-on time. Secondary endpoints included a further performance of CPR and the perceived task load assessed by the NASA task-load index. Results: Hands-on times were lower in PPE teams than in the control group (86% (83–89) vs. 90% (87–93); difference 3, 95% CI for difference 3–4, p < 0.0001). Moreover, PPE teams made fewer change-overs and delayed defibrillation and administration of drugs. PPE teams perceived higher task loads (57 (44–67) vs. 63 (53–71); difference 6, 95% CI for difference 5–8, p < 0.0001) and scored higher in the domains physical and temporal demand, performance, and effort. Leadership allocation had no effect on primary and secondary endpoints. Conclusions: Having to wear PPE during CPR is an additional burden in an already demanding task. PPE is associated with an increase in perceived task load, lower hands-on times, fewer change-overs, and delays in defibrillation and the administration of drugs. (German study register number DRKS00023184). The current European Resuscitation Council COVID-19 guidelines recommend the use of personal protective equipment (PPE) during the cardiopulmonary resuscitation (CPR) of a suspected COVID-19 victim . These rThe beneficial effects of PPE include counteracting aerosol or droplet transmission-induced infection rates ,2,3. DelInvestigating the impact of PPE on the quality of CPR in COVID-19 patients in large randomized trials would be difficult in real cases for a variety of reasons. Simulation allows the investigation of team performance both globally and in specific subtasks in a realistic and standardized manner . A partihttp://www.aim-arnsberg.de; assessed on 19 August 2022), organizes educational courses for physicians, mainly residents in their 2nd to 3rd year of postgraduate medical education in internal medicine, anaesthesia, or surgery, from Germany and German-speaking countries working in intensive care and emergency medicine. Participants of the courses were offered to attend voluntary simulator-based CPR workshops and were informed that simulations were video-taped for scientific reasons. Identical workshops were offered to physicians wishing to participate without being filmed. To capture the actual level of training of the course participants, a specific theoretical instruction on their knowledge of CPR was omitted. The updates in international CPR guidelines  did not affect the primary and secondary outcomes of the present trial. The trial was carried out following the rules of the Declaration of Helsinki and was approved by the Ethics Committee of “Aerztekammer Westfalen-Lippe” (2020-602-f-S) that waived the obligation to obtain consent. The study is registered at the German Clinical Trial Registry  and reported herein according to the extensions to the CONSORT statements of the Reporting Guidelines for Health Care Simulation Research , unless otherwise stated. Statistical analysis was performed using SPSS (version 28). Numerical data were analysed by a non-parametric ANOVA, followed by a Mann–Whitney test, if appropriate. The estimates for differences between the medians and their approximate confidence intervals were obtained by the Hodges–Lehmann estimation. Categorical data were analysed using the chi-square test. A p = 0.31). The PPE teams needed 62 (50–78) s for “donning”.One hundred and ninety-eight PPE teams  were compared with 423 control teams (1451 participants). The gender of the leaders was equally distributed between the control groups and the PPE teams (p < 0.0001). Leadership allocation had no effect on the hands-on times (p = 0.77). For more information, please see The hands-on times were lower in the PPE group than in the control group (86% (83–89) vs. 90% (87–93); difference 3, 95% CI for difference 3–4, The performance metrics of CPR are displayed in p < 0.0001) and scored higher in the domains physical and temporal demand, performance, and effort (p = 0.63) nor on any of the six domains (The PPE teams perceived higher task loads than the control group teams (57 (44–67) vs. 63 (53–71); difference 6, 95% CI for difference 5–8, d effort . Leaders domains .This prospective comparative trial demonstrates that wearing PPE while performing CPR increased the rescuers’ task-load and resulted in lower hands-on times, too few change-overs, and delays in defibrillation and the administration of drugs. Designated leadership did not mitigate the additional burden imposed by PPE.To the best of our knowledge, this is the first large-scale trial on the effects of PPE in CPR teams. Simulator-based cross-over studies from the pre-COVID-19 area reported a significant deterioration in chest compression performance in participants wearing level-C PPE ,13,14. SIn keeping with our findings of increased physical demand, performance, and effort in PPE teams, Chen et al. reported that rescuers’ increases in heart rate, mean arterial pressure, and subjective fatigue scores were significantly more obvious with the use of PPE, which was associated with a significant decrease in effective chest compressions . By contAs far as we know, this is the first trial investigating leadership in the context of CPR teams equipped with PPE. Our results indicate that designated leadership is not able to mitigate the additional burden imposed by PPE.Strengths of this trial include the large sample size and the perfectly identical conditions for all teams. Limitations include the non-randomized design and the absence of real patients. However, randomized trials on PPE in real COVID-19 cases are difficult to conduct. Moreover, simulation is increasingly regarded as an accepted tool for evaluation  and perfOur study population consisted of physicians in their 2nd to 3rd year of residency that, at the time of the study, acted as potential first responders for cardiac arrests in their hospitals. In addition, we refrained from using special teaching, special PPE protocols, or habituation with repetitive exposure prior to testing our participants in the simulated scenario. As such, our results reflect the actual state of our participants’ knowledge and skills and can be extrapolated to real-world settings. Unfortunately, chest compression depth, a benchmark for fatigue during CPR described in previous studies, could not be assessed with our mannequin due to technical limitations.Clinical studies have established that deviations from CPR algorithms are associated with decreased rates of return of spontaneous circulation and survival to discharge ,31. The Our results indicate that training CPR under the conditions which require PPE is warranted and may be a suitable countermeasure against shortcomings and delays. As first responders have to potentially act under various circumstances requiring PPE, PPE sessions should be integrated in regular teaching and training regardless of the current pandemic situation. The participants should be made aware of potential PPE-related increases in rescuers’ fatigue and advised to frequently change-over.Having to wear PPE during CPR is an additional burden for rescuers involved in an already demanding task. PPE is associated with an increase in perceived task load and lower hands-on times, fewer change-overs, and delays in defibrillation and the administration of drugs. These findings should be considered in future resuscitation training.