To prevent infection and thrombosis of central venous catheters (CVCs) in hemodialysis patients, different CVC lock solutions are available. Taurolidine-based solutions and citrate in different concentrations are frequently used, but no definite conclusions with regard to superiority have been drawn.In this retrospective, observational, multicenter study, we aimed to assess the risk for removal of CVC due to infection or catheter malfunction in hemodialysis patients with CVC access for different lock solutions: taurolidine, high-concentrated citrate (46.7%) and low-concentrated citrate (4 or 30%). A multivariable Cox-regression model was used to calculate hazard ratio’s (HR).We identified 1514 patients . In 96 (6%) taurolidine-based lock solutions were used. In 1418 (94%) citrate-based lock solutions were used . Taurolidine-based lock solutions were associated with a significantly lower hazard for removal of CVC due to infection or malfunction combined , and for removal of CVC due to infection or malfunction separately . High-concentrated citrate lock solutions were not associated with a decreased hazard for our outcomes, compared to low-concentrated citrate lock solutions.Removal of CVC due to infection or catheter malfunction occurred less often with taurolidine-based lock solutions. We present the largest cohort comparing taurolidine- and citrate-based lock solutions yet. However, due to the retrospective observational nature of this study, conclusions with regard to superiority should be drawn with caution. In patients with kidney failure, arteriovenous fistulas (AVF) are first choice option for vascular access for hemodialysis . HoweverCentral venous catheters (CVC) provide easy and instant vascular access. However, CVC are associated with an increased risk for infectious complications and catheter malfunction, when compared to AVF . InfectiTo prevent infection and thrombosis, different CVC lock solutions are available and used. In all patients with CVC for hemodialysis, lock solutions are indicated and used. Choice for lock solution type may be determined by the treating physician, since there is no hard evidence on superiority of any of the available options, according to the guidlines . For locAltogether, there is an urgent need for comparison taurolidine- and citrate-based lock solutions in different concentrations. We aimed to evaluate the efficacy of taurolidine- and citrate-based lock solutions in reducing the risk of infectious complications and catheter malfunction in a large cohort of patients with CVC for hemodialysis. Moreover, we describe characteristics and the incidence of CVC-related infections in a large cohort of patients with CVC for hemodialysis.We performed a retrospective observational multicenter cohort study to assess the risk of infection and catheter malfunction during use of taurolidine- and citrate-based lock solutions in hemodialysis patients with CVC access . All patThe primary outcome of this study was the hazard ratio for a combined endpoint consisting removal of CVC due to either infection or due to catheter malfunction. The secondary outcomes of this study were the hazard ratios for (1) removal of CVC due to infection or (2) removal of CVC due to catheter malfunction separately. Only events occurring during the first CVC of each patient were studied in formal analysis, since the chance of events for consecutive CVC may also be dependent on the occurrence of events during earlier CVC. Additional to our primary and secondary outcomes, the incidence and incidence rates of proven CVC-related BSI with corresponding pathogens were reported in the entire cohort. A proven CVC-related BSI was defined as the presence of a positive blood culture, associated with a raised systemic inflammatory response, and absence of clinical signs of a non-CVC-related source of infection.The lock solutions that were studied in this analysis were taurolidine-based lock solutions (consisting of taurolidine combined with citrate 4% and/or 500 U heparin/ml), high-concentrated citrate lock solutions 46.7%) and low-concentrated citrate lock solutions (with either 4% or 30% citrate lock solutions). Choice for lock solution is non-standardized and based on the preference of hemodialysis center and treating physician  [.7% and lP-values ≤0.05 were considered statistically significant. The incidence of BSI was reported for all CVC as number of events per 1000 CVC days. Patients with missing data with regard to the determinant, covariates or outcome were excluded from formal analysis. Numbers of missing data were reported in the results section of this article.Data were stored in an SPSS (version 21.0) database. Descriptive data were generated in SPSS. All statistical analyses were performed in R studio (version 3.2.2). For survival analysis, patients were censored after the first event, since consecutive events within patients are not independent. Multivariable analysis was performed with a Cox proportional hazards model. Cause specific hazard ratios (HR) with 95% confidence intervals (CI) for primary and secondary outcomes were calculated for different lock solutions. On theoretical grounds, age and gender were identified as potential confounders and entered as covariates in the model. No random effects were fitted in the model to correct for correlation in data of patients from the same hospitals, since the lock solution type was strongly correlated to hospital site. The proportional hazards assumption was verified with both formal tests and graphically, using Schoenfeld residuals. The Cox regression models were fitted with the “cmprsk” and “survival” packages. Due to the observational, non-standardized nature of this study, extraneous factors may influence the results, leading to residual confounding. To explore the possibility of such factors explaining our findings, sensitivity analyses were performed. Since most patients using taurolidine-based lock solutions originated from one specific center, the main analysis  was repeated within this specific center only. Also, the analysis was repeated with a frailty term integrated in the model to correct for correlation in data of patients from the same center (assuming a Gaussian distribution of the frailty term). Furthermore, the analysis was repeated for patients with jugular CVC only, to explore if CVC insertion site influenced the outcomes. Also, the analysis was repeated with stratification for CVC type , to explore if CVC type influenced the outcomes of this study. Finally, we studied 30% citrate lock-solutions and 46.7% citrate lock-solutions together (eliminating 4% citrate lock-solutions) for comparison to taurolidine-based lock solutions, to investigate if the definition of our groups influence the outcomes of this study.n = 942) were male. For 640 patients, hemodialysis was initiated in an acute setting (40%). For 1514 patients, the lock solution of the first line consisted of taurolidine in 96 patients , high-concentrated citrate in 1110 patients  and low-concentrated citrate in 308 patients . See Table In total, we identified 1603 unique patients. In 40 patients, heparin-based lock solutions were used. In 49 patients, data with regard to lock solution type were missing. These patients were excluded from analysis, leaving 1514 patients with a total of 139,217 CVC days. Median age for all patients was 65 years (IQR 53–74), 59% of patients  with a HR of 0.34 (95% CI 0.19–0.64). When studying both secondary endpoints separately, taurolidine-based lock solutions were also associated with a significantly lower hazard for removal of CVC due to infection  and removal of CVC due to catheter malfunction  compared to low-concentrated citrate lock solutions (4% or 30%). High-concentrated citrate lock solutions were not associated with a decreased hazard for the primary and secondary outcome compared to low-concentrated citrate lock solutions (Table 2).CVC was removed due to infection in 156 patients  and due to catheter malfunction in 163 patients  with taurolidine- or citrate-based locks. The numbers of events for different lock solutions are summarized in Table 1n = 178), findings were similar to the initial analysis . When studying the hazard for our primary outcome in all patients after correction for center, findings were similar to the initial analysis again . The estimates did not change when studying jugular CVC only  or after stratification for CVC type  . Furthermore, the estimates did not change when regrouping citrate 30 and 46.7% lock solutions together (and eliminating citrate 4% lock solutions), and comparing this group to taurolidine lock solutions .When studying the hazard for our primary outcome within the one center accounting for 94% of patients using taurolidine-based lock solutions  with taurolidine-based and citrate-based lock solutions. In all 1603 patients, 2749 CVC were placed with a total of 271,295 CVC days. Overall incidence rate of proven catheter-related BSI was 0.83 per 1000 CVC days. The incidence rate of proven catheter-related BSI was lowest for taurolidine-based lock solutions (0.37 per 1000 CVC days) compared to citrate-based lock solutions (0.89 per 1000 days for high-concentrated citrate and 0.83 per 1000 days for low-concentrated citrate). More details on causative pathogens are summarized in Table Data with regard to lock solution type were missing in 3% (49 / 1603). Data with regard to the outcomes were missing in < 0.001% (1 / 1603). Data with regard to covariates were missing in < 0.01% (6 / 1603).In this retrospective, observational cohort study, removal of CVC due to infection or catheter malfunction occurred less often in hemodialysis patients with taurolidine-based lock solutions compared to citrate-based solutions. We did not observe any differences between high-concentrated citrate lock solutions and low-concentrated lock solutions. Our findings suggest that taurolidine-based lock solutions may be superior to citrate-based lock solutions in terms of infections and catheter malfunction.In earlier studies, conflicting results were found with regard to the optimal lock solution. It has been established that taurolidine- and citrate-based lock solutions are superior to heparin-based lock solutions in terms of CVC-related BSI and equally effective in prevention of CVC-associated thrombosis –14. HoweBesides the effect of lock solution type, we presented the number of proven CVC-related BSI in our cohort. The CVC-related BSI per 1000 CVC days in our cohort was < 1, which is below the targeted CVC-related BSI rate during hemodialysis in the Netherlands . We obseS. aureus, and slightly more often by gram-negative pathogens. In vitro, both citrate and taurolidine-based lock solutions are active against a large spectrum of gram-negative and gram-positive micro-organisms [Staphylococcal bloodstream infections [It appeared that CVC-related BSI in patients with taurolidine-based locks were less often caused by rganisms , 25. In fections . From thThe major strength of the current study is the large number of patients studied, included from 12 academic and non-academic hospital sites throughout the Netherlands over 5 years. Apart from one small RCT , there aOur study has drawbacks as well. Due to the observational nature of this study, decisions with regard to lock solution type were unstandardized. This may lead to confounding by indication: clinicians may provide more proactive and preventive care to the most vulnerable patients, leading to underestimation of the effect of such a strategy. Second, we observed that lock solution type is depending on hospital site. The choice for lock solution type probably does not depend on patient characteristics but on routine practice, that differs between centers. Such ‘center effect’ might explain part of the lower hazard for our outcomes in patients treated with taurolidine-based locks. Indeed, most patients using taurolidine-based lock solutions were included in one center. In this center, approximately half of patients used taurolidine-based lock solutions, the other half used citrate lock solutions: prior to 2014 patients used citrate lock solutions predominantly, while patients treated after 2014 used taurolidine-based lock solutions predominantly. We studied the effect of lock solution type within this center and after correction for center as a sensitivity analysis, to evaluate if the lower hazard for infection or catheter malfunction remains: results were comparable to the analysis of the entire cohort. If this specific center would have changed their hygiene policy simultaneously with their choice of lock solution, this may results in bias. However, the fact that the use of taurolidine-based lock solutions was associated with both secondary endpoints separately, suggests otherwise . Nevertheless, validation of these findings in a randomized controlled setting is desirable.Moreover, any change of lock solution type within patients during insertion of one CVC, has not been registered in our dataset. This may distort the outcomes of our study. However, since choice of lock solution type is mainly depending on hemodialysis center and treating physician, the number of alteration within patients is probably very low.In conclusion, removal of CVC due to infection or catheter malfunction occurred less often in hemodialysis patients with taurolidine-based lock solutions. High-concentrated citrate lock solutions (46.7%) were not superior to lower concentrated citrate lock solutions (30% or 4%). Due to the retrospective observational nature of this study, definite conclusions with regard to superiority should be drawn with caution: validation of our findings in a prospective and standardized setting, ideally in a randomized controlled trial, is desirable.


Viruses cause a wide spectrum of clinical disease, the majority being acute respiratory infections (ARI). In most cases, ARI symptoms are similar for different viruses although severity can be variable. The objective of this study was to understand the shared and unique elements of the host transcriptional response to different viral pathogens. We identified 162 subjects in the US and Sri Lanka with infections due to influenza, enterovirus/rhinovirus, human metapneumovirus, dengue virus, cytomegalovirus, Epstein Barr Virus, or adenovirus. Our dataset allowed us to identify common pathways at the molecular level as well as virus-specific differences in the host immune response. Conserved elements of the host response to these viral infections highlighted the importance of interferon pathway activation. However, the magnitude of the responses varied between pathogens. We also identified virus-specific responses to influenza, enterovirus/rhinovirus, and dengue infections. Influenza-specific differentially expressed genes (DEG) revealed up-regulation of pathways related to viral defense and down-regulation of pathways related to T cell and neutrophil responses. Functional analysis of entero/rhinovirus-specific DEGs revealed up-regulation of pathways for neutrophil activation, negative regulation of immune response, and p38MAPK cascade and down-regulation of virus defenses and complement activation. Functional analysis of dengue-specific up-regulated DEGs showed enrichment of pathways for DNA replication and cell division whereas down-regulated DEGs were mainly associated with erythrocyte and myeloid cell homeostasis, reactive oxygen and peroxide metabolic processes. In conclusion, our study will contribute to a better understanding of molecular mechanisms to viral infections in humans and the identification of biomarkers to distinguish different types of viral infections. Respiratory viral infections represent a significant threat to human health worldwide. Prior to the SARS-CoV-2 pandemic, influenza was the most common cause of morbidity and mortality in adults with respiratory viral infections . ImplemeHere, we performed transcriptomic analysis from the peripheral blood of patients infected with different viruses including respiratory and non-respiratory infections. We identified conserved elements of the host response common to all included viruses and we identified virus-specific responses. These findings confirm the potential for host response diagnostics to differentiate between viral infections despite their shared clinical features.Patients were enrolled prospectively for sample collection in the Emergency Departments (EDs) of three hospitals from 2009 through 2016: Duke University Medical Center , Durham VA Health Care System , and UNC Health Care . Enrollment was by convenience sampling as part of two consecutively executed observational studies: Community Acquired Pneumonia and Sepsis Study (CAPSS), and Rapid Diagnostics in Categorizing Acute Lung Infection (RADICAL) study. Patients were eligible for CAPSS if they were ≥6-years old with a known or suspected infection of <28-days duration and if they exhibited two or more systemic inflammatory response syndrome criteria . RADICALUpon enrollment, whole blood was collected into PAXgene Blood RNA tubes (Qiagen) and processed according to manufacturer instructions with storage at -80°C. Total RNA was extracted using QIAsymphony PAXgene Blood RNA Kit on QIAsymphony SP instrument (QIAGEN). RNA yield was determined using Qubit RNA BR Assay Kit read on Victor X2 Plate Reader (Perkin Elmer), and quality assessed using Agilent HS RNA (15NT) Kit read on Fragment Analyzer System (Agilent). Strand-specific RNA sequencing libraries were generated using NuGEN Universal Plus mRNA-Seq kit with AnyDeplete-mediated human globin transcript depletion . The libraries were sequenced on Illumina NovaSeq 6000 S4 flow cell at 150bp paired end reads with an average of 50M read pairs per RNA sample.http://www.bioinformatics.babraham.ac.uk/projects/fastqc), then quality trimmed using Trimgalore  with default settings. Trimmed reads were mapped to human genome annotation hg38 (ENSMBL hg38 release 91) using STAR . inc. inc20)]Among the down-regulated DEGs, all three respiratory viruses induced changes that were enriched for protein targeting to membranes and cell differentiation pathways. However, ENV and MPV infections exhibited a stronger down-regulation of T cell activation and cell adhesion molecules compared to IAV infections. Down-regulation of adaptive immune response signals is a common feature of respiratory virus infections that has been described for SARS-CoV-2 , 45, 46 in vivo, resulting in reduced interferon induction and pathway activation  . Adding CCL2 (C-C Motif Chemokine Ligand 2), CCL8 (C-C Motif Chemokine Ligand 8), CXCL10 (C-X-C Motif Chemokine Ligand 10), and DEFB1 (Defensin Beta 1). The first three genes represent cytokine/chemokine genes, which are a central component of the innate immune response. DEFB1 is a member of the defensin gene family that consist of microbicidal and cytotoxic peptides mainly produced by neutrophils. The most down-regulated genes were ARG1 (Arginase 1), OLAH (Oleoyl-ACP Hydrolase), CA4 (Carbonic Anhydrase 4) and CNTNAP3 (Contactin Associated Protein Family Member 3). ARG1 catalyzes the hydrolysis of arginine to ornithine and urea. Arginine metabolism is a critical regulator of innate and adaptive immune responses where it is an antimicrobial effector pathway in polymorphonuclear granulocytes. OLAH contributes to the release of free fatty acids from fatty acid synthase, and CA4 catalyze the reversible hydration of carbon dioxide. Both genes have no known function in the host defense against viral infections. CNTNAP3 represents a cell-recognition molecule mediating glial cell contacts. It has not previously been implicated in in the host response to viral infections.Comparing the host response to IAV infections versus all other respiratory viruses, we identified 374 DEGs. The top four IAV up-regulated genes were CNTNAP3, CNTNAP3B, PI3, and PHF24. Note that CNTNAP3 was downregulated in the IAV-specific response but was up-regulated in ENV infections. CNTNAP3B, like CNTNAP3, is a cell-recognition molecule mediating glial cell contacts and has not been identified as part of the host response to viral infection. PI3 (PHD Finger Protein 24) has no known function and PHF4 (Peptidase Inhibitor 3) represents a neutrophil-specific elastase with anti-microbial functions.We also identified 235 ENV-specific DEGs. The top four up-regulated genes were In contrast to IAV and ENV, we did not identify a MPV-specific signature. One explanation may be that the response to MPV is intermediate to the comparator viruses and therefore, the magnitude of difference between MPV and either IAV or ENV is too small to detect. Alternatively, there may have been too few MPV cases to detect small but significant differences. Future studies with more balanced and larger groups or a meta-analysis including other cohorts may resolve this issue.CXCL10, CMPK2, RSAD2, SERPINA3, and TNFAIP6 were up-regulated whereas CXCL14, IVNS1ABP, and ZMAT3 were down-regulated in comparison to IAV infections. Similar to these findings, in our dataset, IVNS1ABP and ZMAT3 were downregulated in ENV infections compared to IAV infection. In contrast, in our dataset, CXCL10, CMPK2, RSAD2, and TNFAIP6 were significantly down-regulated in ENV infections compared to IAV infections. Thus, the direction of the regulation of genes in adult and pediatric patients may be different for several innate immune response genes. This hypothesis will have to be confirmed in future studies.Our results are in good agreement with similar studies described in the literature. A dataset published by Zhai et al.  reportedBCL2L1 (BCL2 Like 1). BCL2L1 is involved in the regulation of mitochondrial membrane potential and controls production of reactive oxygen species and release of cytochrome C, which are potent inducers of cell apoptosis. Our observations suggest that DENV induces a greater degree of cellular proliferation of circulating immune cells than we observed for respiratory viral infections. This may be due to the systemic nature of dengue, which is not restricted to the respiratory tract. It is also possible that infection of blood lymphocytes by DENV itself initiates the proliferation of blood cells. The increase in pro-B cells, observed in our DCQ, may support this hypothesis. However, more target studies will be necessary to validate this interpretation. Future studies with single cell RNAseq will help address these possibilities. In addition, performing ATACseq on PBMCs of Dengue-infected subjects would be another important future approach to interrogate cell-specific chromatin changes.The host response to dengue infection overlapped considerably with that of other viruses. However, there were also notable DENV-specific responses including an up-regulation of histone-associated and cell cycle regulating genes. An analysis of protein-protein-interaction networks for these DENV-specific DEGs using the STRING interaction database, revealed networks regulated by the CDC6 (Cell Division Cycle 6) protein. CDC6 is essential for the initiation of DNA replication and functions as a regulator at the early steps of DNA replication. Almost all DENV-specific host response changes included an upregulation of gene expression. A notable exception was Our study has several limitations. Most subjects in this study had mild or moderate clinical disease, limiting our ability to identify severity-dependent host changes. It is also possible that subjects with more severe infections, such as due to IAV or DENV, presented to clinical care earlier in the disease course. If so, it is possible that virus-specific differences could have represented different phases of the host response. There is also the possibility that confounding variables contributed to the observed gene expression differences. Most demographic variables were evenly distributed among the groups although this was not universally the case. Analyses to identify such confounding variables revealed age and chronic lung disease may have contributed. Although the viral pathogen itself was the largest contributor to observed differences, we cannot exclude the potential impact of these other variables. Information about duration of symptoms/time of sampling and the potential impact of treatment was not available for most subjects, precluding our ability to control for this. There was an age difference in the comparison groups, with infected being 40s to 60s, and healthy controls being more uniformly between 18-20. Moreover, the Sri Lanka subjects tended to be older than U.S. subjects. However, we did not adjust for age since there was still a significant amount of heterogeneity among ages within each group. We also note that nearly all subjects with DENV came from Sri Lanka, although the Sri Lanka cohort included several other types of infection as well. Nevertheless, we cannot discount the role that genetic variability may have played in these results. Here, bulk RNAseq was used to measure gene expression changes in the blood. Furthermore, analysis of respiratory samples may be more appropriate to study responses in infected tissues. This approach does not identify cell-specific expression changes. Future experiments could perform single cell RNAseq to confirm some of our findings and identify cell type-specific anti-viral host responses. Furthermore, information about the viral genomes  could have identified pathogen-derived factors  responsible for the observed host response differences. Another limitation is that gene expression analyses were performed in blood cells but not in the infected respiratory tract tissues, which may differ . Our stuIn conclusion, we compared the host response to different viral infections in humans and determined common pathways but also virus-specific differences in the host response at the molecular level. Inclusion of multiple types of viruses revealed there to be a pan-viral response. This study contributes to our understanding of the host transcriptional responses to viral infections in humans and the identification of biomarkers that could be used as a diagnostic strategy for pathogen identification. It provides a basis for follow-up validation studies in cell culture and animal models and identifies many genes and pathways as potential targets for future host-targeted drug development.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article text (M&M) and in the All studies involving human participants were reviewed and approved by the Duke University Institutional Review Board, the UNC Institutional Review Board, the Durham VA Institutional Review Board, and the Ethical Review Committee, Faculty of Medicine, University of Ruhuna. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.ET, MM, CW, GG, and KS conceived and designed the experiments. ET, EK, CB, AN, WK, VD, RK, MM, CW, GT, and MR collected the samples. KS and TB performed the experiments. ET, AA, YL, RH, KS, and CF analyzed the data. ET, KS, and CF wrote the manuscript. ET, CW, MM, KS, and GG provided funding. All authors contributed to the article and approved the submitted version.This work was supported by intra-mural grants from the Helmholtz-Association (Program Infection and Immunity), a start-up grant from the University of Memphis Tennessee Health Science Center, NIAID Research Grants 2-U19-AI100625-06 REVISED and 5U19A|100625-07 awarded to KS. This work was also supported in part by the National Institute of Allergy and Infectious Diseases of the National Institute of Health [grant numbers U01AI066569 and UM1AI104681] and the U.S. DARPA [contract number N66001-09-C2082]. The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health. CF was supported by the Eugene A. Stead Scholarship from Duke University School of Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.ELT, GSG, CWW, and TWB consult for and hold equity in Biomeme, Inc.The remaining 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.


MLH1 by NGS, which was confirmed as dMMR by IHC staining. NGS had positive and negative predictive values of 100% (5/5) and 98.7% (74/75), respectively, for MSI-H. The concordance between NGS and PCR-based testing was 98.8% (79/80). Thus, NGS can be useful for evaluating MSI/MMR status in clinical practice and can be an important alternative method for detecting MSI-H/dMMR in the future.Various malignancies exhibit high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR). The MSI-IVD kit, a polymerase chain reaction (PCR)-based method, was the first tumor-agnostic companion diagnostic to detect MSI status in MSI-H solid tumors. Recently, next-generation sequencing (NGS), which can also detect MSI-H/dMMR, has been made clinically available; however, its real-world concordance with PCR-based testing of MSI-H/dMMR remains to be investigated. The co-primary end points included the positive and negative predictive values of MSI-H/dMMR. A retrospective analysis of 80 patients who had undergone both MSI testing and NGS between July 2015 and March 2021 was conducted. Five patients were confirmed to have MSI-H in both examinations. Among the 75 patients diagnosed as microsatellite stable (MSS) by PCR-based testing, one with pancreatic cancer was diagnosed as having MSI-H after NGS. One patient with pancreatic cancer was diagnosed as having MSS in both tests was found to have a mutation in  Approximately 1%–20% of various advanced malignancies exhibited MSI-H/dMMR, which is associated with specific clinicopathological, genomic, or prognostic features6. A deficiency in the mismatch repair pathway has been known to induce microsatellite instability (MSI), an accumulation of DNA replication errors, particularly in genome areas with short repetitive nucleotide sequences. dMMR is indicated by the loss of function of MLH1, MSH2, MSH6, or PMS2 proteins, leading to the loss of function of the mismatch repair pathway, which plays a key role in maintaining genomic stability7. MSI-H/dMMR tumors produce an increasing number of mutations and neoantigens. CD8+ T cells recognize these neoantigens, resulting in immune cell infiltration into tumors higher than microsatellite-stable (MSS) or proficient MMR (pMMR) tumors8. Recently, Le et al. reported that patients with MSI-H/dMMR cancers had robust responses to anti-PD-1 antibody9. Given the promising results, the US Food and Drug Administration (FDA) approved the use of pembrolizumab, a humanized IgG4 monoclonal antibody, for the treatment of patients with unresectable or metastatic MSI-H/dMMR solid tumors10.Recently, immunotherapy has emerged as a new standard of care for patients with various malignancies; however, only few patients have benefited from programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) blockade. High microsatellite instability (MSI-H) and deficient mismatch repair (dMMR) have been among the established biomarkers for predicting response to immunotherapy, regardless of tumor type11. PCR-based testing is used to determine the MSI status using DNA extracted from tumor tissues without using blood samples as reference and to analyze five mononucleotide repeat markers , which are less susceptible to genetic polymorphisms. The lengths of PCR products from normal DNA are almost confined within the quasimonomorphic variation range (QMVR)12. The concordance of PCR-based testing and the standard method using both tumor and normal tissue DNA was evaluated and showed complete consistency13. Studies have suggested the existence of possible differences in microsatellite marker length according to tumor type. Considering that the PCR-based test is used to evaluate each microsatellite marker on the basis of an QMVR width of ± 3 bases, a slight movement in the wave of each microsatellite marker may result in false-negative results in some solid tumors14. Moreover, reports have found differences in the concordance between PCR-based testing and immunohistochemistry (IHC) staining according to tumor types, such as brain tumor, cholangiocarcinoma, ovarian cancer, and endometrial cancer17.The MSI-IVD kit , which can detect MSI status using a polymerase chain reaction (PCR)-based method, was approved in 2018 as the first tumor-agnostic companion diagnostic for pembrolizumab in patients with MSI-H solid tumors18. Although several studies have reported the concordance between PCR-based testing and NGS and suggested a favorable concordance of 99.4%20, only few reports have evaluated the real-world concordance of MSI status in PCR-based testing and NGS using data from clinical practice. Given that MSI-H is an established predictive biomarker for immune checkpoint inhibitors, misdiagnosis might influence the therapeutic strategy for patients who would benefit from immune checkpoint blockade. In Europe and the United States, IHC staining has also been recommended for detecting MSI-H/dMMR, which is backed by clinical trials21. In Japan, however, PCR-based MSI testing has been the only approved companion diagnostic for pembrolizumab. The present study investigated the concordance between PCR-based testing and NGS and determined the usefulness of NGS for evaluating MSI-H/dMMR using real-world data in Japan.Recently, next-generation sequencing (NGS) has emerged as an essential tool not only for detecting genomic alterations indicated for molecular targeted agents but also for precise clinical decision making, including risk assessment, diagnosis, and prognosis. Some NGS platforms, irrespective of the type of the commercial or noncommercial base, can also determine MSI status, dMMR, or both. However, differences in the methods used to determine MSI-H have been found across NGS platforms according to the microsatellite markers adopted and algorithm for deciding the MSI-H used. Discrepancies in MSI status assessed using PCR-based testing, NGS, or dMMR by IHC staining have been observed. While approximately 96% concordance between PCR-based testing and IHC staining has been reported, the correlation between PCR-based testing and NGS is yet to be thoroughly investigatedMSH6 and PMS2. Patients with failed analyses of NGS or PCR-based MSI test results due to any reasons were excluded. We obtained information on the patients’ characteristics from their medical records.Patients with solid tumors who underwent both PCR-based testing and NGS of any platform and were evaluated for their MSI status between July 2015 and May 2020 at Keio University Hospital were retrospectively analyzed. Patients who underwent NGS using the NCC Oncopanel  were excluded, given that this platform could not evaluate MSI status and alterations in 12.Tumor DNA from deparaffinized cells was analyzed using PCR with five monomorphic mononucleotide repeat markers  developed by the Promega MSI analysis system . Tumor tissue of unstained slides (5–10 pieces of undyed 5-μm specimens) with tumor cells ≥ 50% were required for the analysis. Tumors were classified as MSI-H when two or more of the five markers were positive for shifts in the allelic bands, whereas tumors with one unstable marker were classified as MSI-L and those without any positive marker were classified as MSS22. Patients with solid tumors who are unresponsive to the standard of care but are eligible for chemotherapy are candidates for this analysis. The test can also be performed in pediatric patients with cancer or patients with orphan cancers as part of the diagnostic process and for developing treatment strategies on the basis of genomic mutation findings. F1CDx can detect substitutions, insertion and deletion alterations, and copy number alterations across 324 genes, selected gene rearrangements, and tumor mutational burden (TMB) using DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens23. To determine MSI status, 95 intronic homopolymer repeat loci (10–20 bp long in the human reference genome) with adequate coverage in the F1CDx assay were analyzed for length variability and compiled into an overall MSI score via principal components analysis24. Using the 95 loci for each sample, the repeat length was calculated in each read that spanned the locus, and an MSI score was produced. Each sample was assigned a status of MSI-H or MSS, and samples with low coverage (<  × 250  median) were assigned a status of MSI-unknown (detailed information available at https://www.foundationmedicine.com/genomic-testing/foundation-one-cdx). For the analysis, 10 pieces of undyed 4- to 5-μm sections were prepared from the FFPE specimens, and samples with ≥ 20% tumor content were needed.FoundationOne CDx  was approved for use in all solid tumors in 2019 by the Pharmaceuticals and Medical Devices Agency, a regulatory authority in Japan25. Genome annotation and curation for analyzing the sequencing data were performed using an original bioinformatics pipeline called GenomeJack , in which mapping of the NGS reads to the human reference genome (UCSC human genome 19) was performed using the Burrows–Wheeler Aligner26, and the reads were realigned with ABRA27. For identification of single-nucleotide variants (SNVs), SAMtools was used to pile up the sequencing reads, and defective SNVs that showed conflict between pairwise reads were abandoned28. The criteria for mutations were as follows: the noise distributions arising from random sequencing errors were determined. Each mutation was evaluated using a binomial test (p < 0.05) to reduce random sequencing errors. We called somatic mutations by comparing the number of mismatch bases in the tumors with those in the normal controls by using the Fisher exact test (p < 0.001). The copy number of each gene was calculated as the median value of all the sequencing reads covering the target genes and compared with the median value of the control samples. In calling copy number alterations (CNAs), we defined more than three-fold copy number increases as “gain” and less than two-fold decreases as “loss.” We identified cancer-specific somatic gene alterations, such as SNVs, insertions/deletions (Indels), and CNAs. Moreover, TMB was measured as a potential biomarker of immunotherapy. In our test, TMB was defined as the number of nonsynonymous and synonymous mutations in the target regions per megabase of tumor genome , and high TMB was defined as at least 10 mutations per megabase (≥ 10 mut/Mb). All the detected gene alterations in 160 cancer-related genes were annotated and curated using the COSMIC (https://cancer.sanger.ac.uk/cosmic), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), CIViC (https://civicdb.org/home), SnpEff24, and Clinical Knowledgebase (CKB) databases (https://ckb.jax.org/). This sequencing system evaluated MSI-H/MSS using the MSIsensor program, which reports the percentage of unstable microsatellites as a score29. In PleSSision, MSIsensor scores ≥ 20 and < 20 are defined as MSI-H and MSS, respectively. For the analysis, 5 pieces of undyed 10-μm sections were prepared from FFPE specimens, and samples with ≥ 20% tumor content were required. Generally, FFPE specimens extracted within 3 years were eligible for evaluation of PCR-based MSI testing and NGS in the present study.PleSSision , an outsourcing clinical sequencing system, allows for targeted amplicon exome sequencing of 160 cancer-related genes using the Illumina MiSeq sequencing platform The tumor tissues that were categorized as MSI-H by PCR-based testing or NGS were analyzed using IHC. The processed IHC slides were evaluated by two pathologists. Cases with loss of at least one expression of MLH1, MSH2, MSH6, or PMS2 in tumor cells were defined as dMMR. Considering the immunostaining topographic heterogeneity, the IHC results of the MSH2/MSH6 and MLH1/PMS2 patterns were confirmed because MMR proteins function as heterodimers. pMMR was defined as a positive nuclear staining of all MMR proteins.κ correlation coefficient (κ) with its 95% CI, maximum value κ (κmax) given the observed distribution, and exact p value. All statistical analyses were performed using the JMP version 14.2.0 software .The positive predictive value of NGS compared with PCR-based testing was calculated as the proportion of the number of patients who were categorized as MSI-H by NGS divided by the number of patients who were considered as MSI-H by PCR-based testing. The negative predictive value of NGS compared with PCR-based testing was calculated as the proportion of the number of patients who were categorized as MSS by NGS divided by the number of patients who were considered as MSI-L/MSS by PCR-based testing. Concordance between NGS and PCR-based testing was calculated as the proportion of the number of patients with MSI-H or MSI-L/MSS categorized by both NGS and PCR-based testing divided by the number of patients. The 95% confidence interval (CI) for the binomial proportion was calculated on the basis of the exact binomial distribution. The frequency and percentage of tumor samples with concordant and discordant MMR statuses based on PCR-based testing and NGS results were calculated, after which the extent of concordance was tested using the Cohen This study was approved by the Keio University Hospital Institutional Ethics Committee  and was performed in accordance with the Declaration of Helsinki and Ethical Guidelines for Medical and Health Research Involving Human Subjects. Written informed consent was obtained from all patients.Between July 2015 and March 2021, 933 patients received any modalities of MSI testing, in which 411 and 379 patients underwent NGS or PCR-based MSI testing only, respectively. After excluding 63 patients who were evaluated using the NCC Oncopanel, 80 patients were ultimately included in the present study. A consort flow diagram is presented in Fig. Among the 80 included patients, 5 (5%) with gastric neuroendocrine carcinoma (50-year-old man), ovarian cancer (60-year-old woman), cancer of unknown primary (40-year-old man), endometrial carcinoma (73-year-old woman), and extramammary Paget’s disease (80-year-old man), respectively, were determined as having MSI-H by both PCR-based testing and NGS.MSH2 mutation in the patient, who was also evaluated with IHC staining, which confirmed dMMR (loss of MSH2 and MSH6). She had already been diagnosed with Lynch syndrome and had a family history of cancer.Of the 75 patients who were initially categorized as having MSI-L/MSS by PCR-based testing, a 47-year-old woman with pancreatic ductal carcinoma was confirmed as having MSI-H and dMMR by NGS  and 74/75 , respectively. Meanwhile, the concordance rate between PCR-based testing and NGS was 79/80  was determined as having MSS in both tests; NGS detected an MLH1 mutation, and IHC staining confirmed dMMR in the patient. MSI status itself is recognized as a surrogate marker of dMMR, whereas the true biomarker for predicting the efficacy of anti-PD-1 antibody is MMR deficiency. Although PCR-based testing could only evaluate MSI status, NGS could analyze both MSI status and gene alterations in MMR genes, which we consider as the strong advantage of NGS. Indeed, NGS permits parallel high-throughput sequencing of a high number of microsatellites and genes and may consequently identify MSI, TMB, and other targetable gene alterations.This retrospective study suggests that owing to its sensitivity and specificity, NGS could be considered an alternative to PCR-based MSI testing, the approved companion diagnostic for detecting MSI-H in Japan. To the best of our knowledge, this is the first study to evaluate the real-world concordance and discordance between PCR-based testing and NGS using clinical data in Japan. The six patients included in the study were identified with MSI-H by PCR-based testing or NGS. Among the patients, one (case 3) was initially determined as having MSS by PCR-based testing and subsequently reevaluated as having MSI-H by NGS. Notably, a false-negative result in PCR-based MSI testing was suspected in this case. Generally, reports have shown that the low tumor burden or increase in tumor DNA degradation over time observed in the samples could be attributed to the false-negative results. The PCR-based MSI testing requires at least a tumor content of 50% in tumor samples. Moreover, several studies have reported that Lynch syndrome with germline mutations in 32 and 46% in PleSSision25. Although the present study was not aimed at comparing these two assays, we consider that they are equivalent in the evaluation of MSI status and detection of MMR deficiency.The present study included two NGS assays (FoundationOne CDx and PleSSision) in which the detection method of gene alterations is different. FoundationOne CDx evaluates only tumor cells, whereas PleSSision collects peripheral blood mononuclear cells from patients as control. The germline variants are also evaluable in PleSSision, which could help determine mismatch repair gene alterations as pathogenic germline variants. The detection method of MSI in each assay is also different. In FoundationOne CDx, 95 intronic homopolymer repeat loci with adequate coverage in the FoundationOne CDx assay are analyzed for length variability and compiled into an overall MSI score via principal components analysis. PleSSision uses an MSIsensor program for evaluating MSI status, which reports the percentage of unstable microsatellites as a score. Furthermore, the number of targeted genes differs, with 324 genes in FoundationOne CDx and 160 genes in PleSSision, respectively. However, these two NGS assays can evaluate gene alterations in four MMR genes. The detection rate of actionable gene alterations in each NGS assay is 62.9% in F1CDx19. Moreover, Middha et al. validated NGS against PCR-based testing and MMR IHC for 138 colorectal cancers (CRCs), including 24 MSI-H CRCs, and 40 uterine endometrioid cancers (UECs), including 15 MSI-H UECs, and showed a concordance of 99.4%20. Although these studies reported concordance between MSI status assessed by NGS and PCR-based testing with a larger number of patients in a laboratory setting, our present study demonstrated the clinical usefulness of NGS to analyze MSI status using real-world data in Japan, where PCR-based MSI testing is the only approved companion diagnostic for the use of immune checkpoint blockade as of now. Furthermore, our study was performed in a situation close to real clinical practice where tissue samples are not abundant or are inadequate for MSI testing analysis, unlike previous reports in which tissue samples were obtained from another study.Our study highlights the accuracy of PCR-based MSI testing and the possible usefulness of NGS in clinical practice. Vanderwalde et al. assessed the concordance between PCR-based testing and NGS in 2189 matched cases of 26 cancer types, including 23 cases with MSI-H. In MSI-H detection, NGS had a sensitivity of 95.8% , specificity of 99.4% , positive predictive value of 94.5% , and negative predictive value of 99.2% , compared with PCR-based testingNGS can be used to investigate a large variety of gene alterations at one time. Generally, PCR-based MSI testing requires tumor specimens with FFPE block or 5–10 pieces of undyed 5-μm pathological specimens with tumor cells ≥ 50%, which is equivalent or large compared with specimens required for NGS to perform genomic testing, which need 5–10 pieces of undyed 5-μm sections with tumor cells ≥ 20%. The number of tumor samples was either limited, particularly in the patients with pancreatic carcinoma, prostate cancer, or cancer of unknown primary, whose tumor burden is generally low or tumor is difficult to access, such as those obtained through biopsy. Hence, we often experienced problems related to the inability to order new genomic testing kits given the lack of available specimens. However, rebiopsy might be difficult for patients with poor conditions or without superficial metastasis. To address such problems, using NGS initially might be an ideal alternative.MLH1, MSH2, MSH6, and PMS225. Furthermore, certain types of NGS can also analyze germline mutations associated with Lynch syndrome by assessing matched normal-tumor pairs33. Although the current NGS assay could not analyze loss of function due to DNA methylation, such an approach could be realized in the near future34. Furthermore, by counting thousands of fractional mutations, NGS is able to evaluate TMB. TMB is known to be one of the novel predictive biomarkers for the efficacy of immunotherapy36. Despite the current lack of approval for the use of pembrolizumab in these populations in Japan, the FDA has approved the use of pembrolizumab for patients with unresectable or metastatic TMB-H (≥ 10 mut/Mb) solid tumors by F1CDx in 2020. Although the cutoff value of TMB-H still remains controversial, clinicians would greatly benefit from knowing TMB values when considering immunotherapy.One of the advantages of NGS is its ability to simultaneously detect gene alterations in Conversely, NGS has some problems that must be considered. First, NGS costs approximately 560,000 yen (5300 US dollars), whereas PCR-based MSI testing costs 20,000 yen (190 US dollars) under the health insurance system in Japan, placing considerable economic burden on the national insurance system. Second, the average turnaround time (TAT) for NGS is approximately 4–5 weeks after sample receipt in the laboratory, which might affect treatment decision making. Furthermore, an expert panel discussion is needed to confirm whether gene alterations are meaningful after results are released.37. Through liquid biopsy, the MSI/MMR status can be determined more quickly and effortlessly, allowing not only the administration of immunotherapy for patients with MSI-H/dMMR tumors but also the recruitment of patients with certain gene alterations in clinical trials38. We believe that NGS should be performed diligently in patients suspected to have MSI-H/dMMR after considering their clinical course, past medical history, and family history, regardless of whether they have been determined as having MSS by PCR-based testing.The usefulness of NGS, including the identification of MSI-H/dMMR status, could supersede that of PCR-based MSI testing, provided that cost reductions and shortening of TAT are addressed. Furthermore, the ability of NGS to be performed earlier and at a more adequate timing has made it an indispensable diagnostic strategy. Moreover, forthcoming advancement would allow MSI-H/dMMR to be evaluated using cell-free DNA from a patient’s blood sample. Accordingly, FoundationOne Liquid CDx  and Guardant360 CDx , which are types of novel platforms for liquid biopsy, can detect MSI status and were approved by the FDA in 202025. Similarly, the sequencing success rate of F1CDx in clinical practice has been reported to be approximately 97%22.Some limitations of the present study inherent to its retrospective nature are as follows: first, this was a single-center study; the sample size was limited with potential bias in patient selection. However, we believe that determining the concordance between NGS and PCR-based MSI testing using data from clinical practice is crucial. Second, we did not usually perform IHC staining in all the cases determined as MSS by PCR-based testing, potentially underestimating the number of patients with dMMR. However, the percentage of MSI-H/dMMR cases in our study was 10%, comparable with those reported in previous reports. Furthermore, the facilities where NGS can be performed are limited, and the conditions for insurance coverage are strictly limited in Japan. Patients with good performance status who have completed the standard treatment or have rare cancers for which a standard treatment has not been established yet are candidates for NGS. Thus, the patients who were included in this study might have been biased, particularly with respect to tumor types. Fourth, the information about the sequencing process of NGS was limited; thus, the success rate of NGS was not calculated in the present study. We aimed to investigate the concordance between the PCR- and NGS-based MSI tests; thus, only the patients who underwent both tests were enrolled in the study. In a previous study, we reported a 99% success rate for CLHURC (the previous version of PleSSision)In conclusion, NGS has comparable usefulness with PCR-based MSI testing for evaluating MSI/MMR status. Thus, NGS can be expected to become an important alternative method for detecting MSI-H/dMMR in the near future after reducing costs, shortening TAT, and improving accessibility at appropriate testing periods.Supplementary Information.


Background: The COVID-19 lockdown increases psychological problems in children and adolescents from the general population. Here we investigate the mental and social health during the COVID-19 lockdown in children and adolescents with pre-existing mental or somatic problems.Methods: We included participants (8–18 years) from a psychiatric (N = 249) and pediatric (N = 90) sample, and compared them to a general population sample (N = 844). Measures were assessed during the first lockdown (April-May 2020) in the Netherlands. Main outcome measures were Patient-Reported Outcomes Measurement Information System (PROMIS®) domains: Global Health, Peer Relationships, Anxiety, Depressive Symptoms, Anger, and Sleep-Related Impairment, as reported by children and youth. Additionally, socio-demographic variables, COVID-19-related questions, changes in atmosphere at home from a parent and child perspective, and children's experiences of lockdown regulations were reported by parents.Results: On all measures except Global Health, the pediatric sample reported least problems. The psychiatric sample reported significantly more problems than the general population sample on all measures except for Anxiety and Peer Relationships. Having a COVID-19 affected friend/relative and a COVID-19 related change in parental work situation negatively moderated outcome, but not in the samples with pre-existing problems. All parents reported significant decreases in atmosphere at home, as did children from the general population.Conclusion: We observed significant differences in mental and social health between three child and adolescent samples during the COVID-19 pandemic lockdown and identified COVID-19-related factors influencing mental and social health. It is well-known that large scale disasters have great impact on the well-being of the general population. Whether human-induced , natural , or technological , disasters are accompanied by an increase in a wide scale of mental and behavioral disorders . PandemiEmpirical studies on mental and social health during the COVID-19 pandemic in children and adolescents are limited , though The current study extends these efforts by investigating mental and social health in vulnerable populations, namely children and adolescents with pre-existing mental problems or with a (chronic) somatic condition, during the first Dutch COVID-19 lockdown. During this lockdown, schools, leisure facilities, bars, and restaurants were closed, people were strongly advised to work from home as much as possible and to comply to social distancing, and it was not allowed to be outside with more than two people unless they were from the same household.Children with mental problems and chronic somatic conditions are known to be at risk for psychosocial problems. In general, they rate their well-being, emotional and social functioning, and psychosocial health lower than their healthy peers –13. The As of yet, few empirical studies have investigated the mental and social effects of COVID-19 related regulations in children with pre-existing mental problems (psychiatric population) and results are mixed. One study in children with autism spectrum disorder (ASD) reported increases in irritability, hyperactivity, inappropriate speech, and speech quality from before to during the pandemic . AnotherThe literature on the consequences of the COVID-19 lockdown in children with a (chronic) somatic condition (pediatric population) is also limited. A recent study has shown that psychosocial stress in children and adolescents with cancer did not increase during the first months of the COVID-19 pandemic in the Netherlands . AdditioThe aim of the current study was to assess the mental and social health of a broad population of children with pre-existing conditions (psychiatric or pediatric) during the first COVID-19 lockdown in the Netherlands and to compare their global health, peer relationships, symptom levels of anxiety, depression and anger, and sleep-related impairment to the general population of children and adolescents. We examined potential associated factors, including COVID-19 specific factors such as having an affected friend or relative. In addition, we described changes in home atmosphere due to the first Dutch COVID-19 lockdown and report qualitative data on children's experiences of the COVID-19 regulations.On March 15th 2020, the first COVID-19 lockdown came into practice in the Netherlands. From that moment on, all schools and child day-care facilities were closed , as well as hairdressers, sport and leisure facilities, bars, and restaurants. Adults were advised to work from home as much as possible. People over the age of 12 were advised to keep social distance (1.5 m), to take hygiene measures , and traveling was discouraged. However, it was still permitted to go outside by oneself, to receive three guests at home, and young children (<12 years) could play outside with their friends. From April 28th 2020, young children (<12 years) were again allowed to play sports outside in a team. On May 11th 2020, were partially reopened, followed by the secondary schools on June 2nd 2020.All families were invited to participate during the first COVID-19 lockdown in The Netherlands (between the end of April and early May 2020). During our measurement window, no changes (except for team sports for children <12 years) in Dutch COVID-19 regulations occurred. Parents were approached via email and children were subsequently approached by their parents. If parents and children were willing to participate, the parents completed a sociodemographic questionnaire and COVID-19-related questions online. The children also completed online COVID-19-related questions and several validated Patient-Reported Outcome Measures (PROMs). We collected data through research websites of the KLIK PROM portal developed specifically for each group. The completion time for children and parents together was ~15 min. All children and parents provided informed consent and the study was approved by the Medical Ethics Committee of the Amsterdam UMC.We included independent samples of children and adolescents (8–18 years) from child and adolescent psychiatric centers and a children's hospital and compared these to a sample from the general population.The psychiatric sample consists of children receiving psychiatric care for varying problems  at one of four tertiary academic child and adolescent psychiatric centers in the Netherlands: Accare, Levvel, Curium, and Karakter. The four centers cover child and adolescent psychiatric care in the northern, western, and eastern part of the Netherlands. We approached parents through email between April 30th and May 12th, 2020. In total, we invited 5,615 children and their parents to participate (response rate 4.4%).The pediatric sample consists of children with a (chronic) somatic condition  who are under treatment in the Emma Children's Hospital Amsterdam UMC. Children who already complete PROMs as standard part of their care were included. We approached parents via email in the first week of May 2020. In total, we invited 1,150 children and their parents to participate (response rate 7.3%).For the general population sample, data were collected via an independent online research agency “Panel Inzicht” in April 2020 [see  for addiThe socio-demographic questionnaire was completed by parents and comprised questions about themselves  and their child . The COVID-19-related questions for parents concerned whether the parent or partner reported a negative change in parental work situation due to COVID-19  and whether or not a friend or relative was infected with COVID-19. Additionally, parents completed two questions regarding the atmosphere at home before and during the first Dutch COVID-19 lockdown on a visual analog scale (range 0–100): “How did you experience the atmosphere at home before the Corona regulations?” and “How do you experience the atmosphere at home now?”The COVID-19-related questions for children also assessed the atmosphere at home: “How did you experience atmosphere at home before the schools were closed?” and “How do you experience the atmosphere at home now?” Finally, we asked an open-ended question: “How are the Corona-regulations for you?”We administered six Dutch Patient-Reported Outcomes Measurement Information System (PROMIS®) pediatric measures (8–18 years) to children and youth: Scale V1.0—Global health  , CAT V2.We used the Statistical Package for Social Sciences (SPSS) version 26.0 for statistical analyses. First, we characterized participants of each sample using descriptive analysis (means and percentages).corrected post-hoc t-tests to determine which samples differed significantly from each other. Next, we tested whether there were differences in proportions of children reporting severe symptoms or poor functioning between samples. “Severe” symptoms and “poor” functioning were defined as 1.5 SD above or below the mean T-score of an independent and representative pre-COVID-19 Dutch sample .Second, we performed analyses of covariance (ANCOVA) to assess differences in PROMIS outcome measures between samples. We corrected for age, sex, and parental country of birth as these are characteristics known to influence mental and social outcomes , 34, 35.h sample , 37, excThird, to determine which variables were associated with the PROMIS measures during COVID-19, we repeated the ANCOVA over all groups and included pertinent independent variables: marital status, region, number of children in the family, parental educational level, change in parental work situation due to COVID-19, and infected relative/friend with COVID-19. As the proportion of the participants from the general population is high, we repeated the ANCOVA without the general population sample to explore whether effects are mainly driven by this group.T-tests in each group to investigate changes in atmosphere at home from before to during COVID-19 as reported by children and parents. In addition, we performed an ANCOVA to assess whether the changes in atmosphere differed between samples, correcting for age and sex.Fourth, we performed paired samples Finally, two authors qualitatively analyzed the children's answers to the open-ended question “How are the corona-regulations for you?” using the method for thematic analysis in psychology . AnswersSociodemographic characteristics of all samples are presented in In the pediatric sample 90 children participated (response rate: 7.3%). The mean age was 12.9 years, 55.6% was male, 33.3% had an infected relative/friend, and 21.1% of their parents had a negative change in parental work situation due to COVID-19.In the general population sample 844 children participated (response rate: 8.4%). The mean age was 13.4 years, 47.4% was male, 23.7% had an infected relative/friend, and 26.2% of their parents experienced a negative change in parental work situation due to COVID-19.p < 0.01, η2 = 0.03–0.04) after controlling for the covariates age, sex, and parental country of birth  on the Global Health , Depressive Symptoms , Anger , and Sleep-Related Impairment  measures.We found significant differences on all self-reported PROMIS measures between the three groups (irth see . The psyp < 0.05) on Peer Relationships , Anxiety , Depressive Symptoms , Anger , and Sleep-Related Impairment .The pediatric sample reported significantly better scores than the general population sample (p < 0.05) on all six PROMIS domains: Global Health , Peer Relationships , Anxiety , Depressive Symptoms , Anger , and Sleep-Related Impairment .The psychiatric sample reported significantly worse scores than the pediatric sample  and lowest in the pediatric sample  with lower Global Health , higher Anxiety , and lower Anger . Male gender was significantly associated with higher Global Health , lower Peer Relationships , lower Anxiety , lower Depressive Symptoms , and lower Sleep–related Impairment . Living in the northern region of the Netherlands was significantly (p < 0.05) associated with lower scores on Depressive Symptoms . Being from a single–parent family was significantly associated (p < 0.05) with lower Global health  and higher Anxiety . Having three children or more within the household was significantly associated (p < 0.05) with higher Anger . For each outcome measure, the group effect remained.In p < 0.01) with more Anxiety , more Depressive Symptoms , more Anger , and more Sleep-Related Impairment . A parent having a friend or relative infected with COVID-19 was significantly associated (p < 0.01) with a higher Anxiety score .Regarding COVID-19-specific variables, a negative change in parental work situation was significantly associated (p < 0.05) with lower Global Health  and lower Anger . Male gender was significantly associated (p < 0.05) with higher Global Health , and lower Depressive Symptoms . We found no associations between COVID-19-specific variables and the social and mental health outcomes in the psychiatric group.Finally, we performed the ANCOVA analyses again in the psychiatric and pediatric groups separately to explore whether effects are driven mainly by the general population sample. In the psychiatric group, we found higher age to be significantly associated (p < 0.05) with higher Sleep-Related Impairment . Male gender was significantly associated (p < 0.05) with lower Depressive Symptoms . A parent having a friend or relative infected with COVID-19 was significantly associated (p < 0.05) with lower Peer Relationships .In the pediatric group, we found higher age to be significantly associated (p < 0.001) at home during the COVID-19 lockdown. All parents reported a negative change in atmosphere  and parents , corrected for age and gender.Children from the psychiatric and pediatric samples reported no differences in atmosphere at home between before (measured retrospectively) and during the COVID-19 lockdown, whereas children in the general population reported a worse atmosphere  indicated that the COVID-19-regulations negatively impacted their daily life. The main problems they experienced (≥5% of the children per group) were missing contact with friends (~42%), not being allowed to go to school (~22%), missing freedom (~16%), not being allowed to participate in sports (~14%), missing joyful activities such as birthdays, shopping, day trips, and (graduation) parties (~13%), and missing extended family . However, there were some small differences between the experiences of the samples. Only children and adolescents from the pediatric and psychiatric sample reported difficulties with keeping social distance , whereas the general population sample was the only group that mentioned boredom due to the COVID-19-regulations. In addition, only children from the psychiatric sample indicated that the  COVID-19-measures made them angry, sad, or insecure. A minority of children reported that their daily life was not affected by COVID-19-regulations (5–11%) or reported that the COVID-19-regulations positively influenced their daily life . See In this study, we compared the mental and social health during the first Dutch COVID-19 pandemic lockdown in the spring of 2020 of children and adolescents with pre-existing psychiatric and somatic problems to a general population sample. We assessed global health, peer relationships, anxiety, depressive symptoms, anger, and sleep-related impairment. Mental and social health were worst for children with pre-existing psychiatric problems, whereas children from the pediatric sample showed the most favorable results.As expected, children with pre-existing psychiatric problems showed most problems in mental and social health. The closing of schools and other services during the COVID-19 pandemic lockdown likely affects this group of children especially since these are places where vulnerable children often seek help first . FurtherIn contrast to the psychiatric sample, children, and adolescents from the pediatric sample showed the least problems on all mental and social health scales. As this population generally suffers from more psychosocial problems than the general population , 12, thiConcerning COVID-19-specific variables, a negative change in parental work situation and having an infected friend or relative contributed to various mental and social health problems  in the general population [see  for resuchange in mental and social health of children and adolescents due to the COVID-19 pandemic lockdown. Finally, there may be individual differences in how children respond to the lockdown. The literature so far hints at a potential diverse range of effects that the COVID-19 pandemic has on children and adolescents with pre-existing vulnerabilities, ranging from an increase in mental health problems to reduction of stress, loss of social pressure, and improved well-being , have agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.JB has served as a member of the advisory board of Medice, Roche, Servier, Angelini and has received speaker fee from Takeda and Medice. The remaining 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.


Objective: The optimal treatment modality for retrograde type A intramural hematoma (IMH) remains debatable. This study evaluated and compared surgical outcomes and aortic remodeling after open aortic repair and thoracic endovascular aortic repair (TEVAR) in patients with retrograde type A IMH with a primary intimal tear or ulcer like projection in the descending aorta.Methods: A single center, retrospective observational study was performed on patients with retrograde type A IMH undergoing either open aortic repair and TEVAR. From June 2009 and November 2019, 46 patients with retrograde type A IMH who received either open aortic repair or TEVAR at our institution were reviewed for clinical outcomes, including post-operative mortality/morbidity, re-intervention rate and aortic remodeling.Results: 33 patients underwent open aortic repair and 13 underwent TEVAR. Median age was 68 years (interquartile range [IQR] 15.2 years) and 63 years (IQR 22.5 years) for the open repair group and TEVAR group, respectively. The median duration of follow-up for TEVAR patients was 37.6 months and 40.3 months for open aortic repair. No difference in the 5-year estimated freedom from all-cause mortality , re-intervention , and aortic-related mortality  were observed between the TEVAR and open repair group, respectively; however, the open repair group had a significantly higher 30-day composite morbidity . All patients from both treatment groups had complete resolution of the IMH in the ascending aorta. With regard to the descending thoracic aorta, TEVAR group had a significantly greater regression in the diameter of the false lumen or IMH thickness when compared to the open repair group .Conclusion: TEVAR and open aortic repair were both effective treatments for retrograde type A IMH, in which no residual ascending aortic IMH was observed during follow-up. TEVAR was also associated with lower post-operative composite morbidities and better descending aortic remodeling. In selected patients with retrograde type A IMH, TEVAR might be a safe, effective alternative treatment modality. Type A intramural hematoma (IMH) is defined by the presence of hemorrhage within the wall of the ascending aorta (AA) in the absence of an intimal flap or false lumen (FL) , 2. SeveA diagnostic search from our institutional integrated database was carried out to filter out patients with a diagnosis of aortic dissection or IMH. Individual chart and radiological image review were then performed to identify those with retrograde type A IMH with primary IT or ULP located in the DTA who had received treatment at our institution between June 2009 and November 2019 . PatientThe diagnoses were established based on specific radiological features observed on contrast-enhanced computed tomography (CT) and transthoracic echocardiography (TTE) or trans-esophageal echocardiography (TEE). The definition of retrograde type A IMH was defined as “the presence of a crescentic or circular high-attenuation area in the AA without contrast enhancement on contrast CT in either the arterial or venous phase, with typical type B aortic dissection (TBAD) or IMH presenting as primary IT or ULP in the DTA” . TTE or Demographics, underlying comorbidities, outcomes and follow-up data were retrieved from our integrated medical database, electronic medical records, and through telephone interview. Measured outcomes included 30-day mortality/composite morbidity, post-operative aortic remodeling and freedom from all-cause mortality/aortic-related mortality/re-intervention. The composite end point of morbidity was defined as the composite of the following: respiratory failure requiring ventilator support for more than 48 h post-operatively, renal failure needing renal replacement therapy, cerebral vascular event, spinal cord ischemia, cardiogenic shock, surgical wound infection and any procedural related adverse events that lead to further intervention. For aortic-related mortality, it was defined as death from either aortic rupture, mal-perfusion, or aortic dissection.Additionally, pre-operative and post-operative CT images were examined to evaluate the aortic morphology and remodeling of the aortic wall. The aortic remodeling was evaluated by measuring the pre-operative and post-operative maximum aortic diameters of the ascending and descending aorta, IMH thickness of the AA, and FL diameter or IMH thickness in the descending thoracic aorta at the level of pulmonary artery bifurcation in the short axis view of contrast-enhanced CT using the TeraRecon System .This study was approved by the institutional review board of National Taiwan University Hospital (NTUH – 202101046RINA) and the board waived the need for informed consent.A suitable candidate for TEVAR should fulfill the followings: (1) absence of severe aortic regurgitation (≧grade 3) (2) no evidence of coronary or cerebral ischemia (3) favorable anatomy of the femoral and iliac arteries. The techniques for TEVAR have been described previously . In brieMidline sternotomy was performed and cardiopulmonary bypass (CPB) was commenced after cannulating the right axillary artery, common femoral artery, right atrium and common femoral vein, with systemic cooling down to 25 ~ 28°C. After direct antegrade cardioplegia infusion, moderate hypothermic arrest was initiated and the innominate artery was clamped, followed by antegrade cerebral perfusion via an 8mm side-graft sewn to the right axillary artery and left common carotid artery cannulation . AA/hemiarch/total arch replacement was then performed firstly by completing the distal anastomosis with or without debranching of the supra-aortic vessels. Upon completion of the distal anastomosis, systemic perfusion was reinstituted through the right axillary/femoral artery and rewarming was initiated. The exact extent of the repair was at the discretion of the operating surgeon, where in some patients total arch/hemiarch replacement was performed. In some patients, antegrade TEVAR under direct vision was selectively performed to address the DTA pathology. Lastly, the proximal anastomosis was completed with the use of Teflon felt.Post-operatively, follow-up CT image was scheduled at 1 week, 3-months, and 6- months after the intervention and annually thereafter. In order to assess aortic remodeling, the following parameters were measured and compared with the baseline pre-operative images: (1) the maximal diameters of the AA and DTA (2) thickness of the IMH in the AA (3) diameter of the FL or thickness of the IMH in the DTA at the level of pulmonary artery bifurcation. Due to the relatively small case numbers, prolonged study period over 13 years and non-consecutive enrollment with variable follow-up period, only the most recent post-operative CT images were used for analysis. Follow-up clinical data including the 30-day in-hospital mortality, aortic related mortality and long-term survival were obtained from following sources: medical charts, outpatient appointment, telephone interview and the institutional integrated medical database.T-test. Continuous variables with a skewed distribution were displayed as median and 25–75% interquartile range (IQR) and were analyzed using Mann-Whitney test and Kruskal-Wallis test. The Conover test was used as a post-hoc test when the p-value for Kruskal-Wallis test was <0.05. Categorical variables were presented as numbers and percentage, and were examined using Fisher's exact test. The all-cause mortality, freedom from aortic related event and freedom from re-intervention were modeled using Kaplan-Meier method, and the log-rank test was applied to test for statistical significance. A p < 0.05 was considered statistically significant. All statistical analysis was performed using MedCalc statistical software version 19.5.3 .Continuous variables that were normally distributed were displayed as mean ± standard deviation (SD) and analyzed using student This study included a total of 46 patients with retrograde type A IMH. 13 received TEVAR and 33 received open repair. The demographics and distribution of our cohort for each surgical approach over the study period are displayed in All patients had CT prior to intervention for the diagnosis and evaluation of aortic morphology . For theThe procedural details for TEVAR were displayed in The operative details for open repair were shown in p = 0.34), re-intervention , and aortic-related mortality  were observed between the TEVAR and open repair group, respectively (The median follow-up duration was 37.57 months (IQR 29.75) for the TEVAR group and 40.33 months (IQR 67.74) for the open repair group. 30-day composite morbidity was higher in patients after open repair, in comparison to TEVAR [13/33 (39.39%) vs. 1/13 (7.69%)]. Stroke, post-operative bleeding requiring re-exploration, renal failure and myocardial stunning were the most common comorbidities after open repair . 30-day ectively .The post-operative CT parameters of the aorta were also displayed in In this study, TEVAR and open aortic repair were both effective in promoting AA remodeling, and TEVAR was associated with lower post-operative composite morbidity and better remodeling of the DTA.A recently published multi-center study had demonstrated that TEVAR is a safe alternative to open repair, with no 30-day mortality and a low re-intervention rate of only 6%. Post-operative neurological complications developed in two (11.1%) patients and renal/respiratory failure in one (5.6%) , and theAlthough more re-interventions were required after TEVAR repair within 3-year of follow-up, these procedures were all simple TEVAR extension in the distal thoracic aorta for distal SINE. Neither retrograde type A aortic dissection nor late aortic related deaths was observed. In the previous report, it was emphasized that patient selection and pre-operative surgical planning were all crucial for the success of TEVAR . ImportaFor aortic remodeling, both open repair and TEVAR could promote AA remodeling by reducing the AA diameter, as well as achieving complete resolution of the IMH. Evidence gathered from studies of TBAD have demonstrated that aortic remodeling was a positive prognostic factor after TEVAR, and was associated with a reduced risk of aortic related complications , and might therefore improve long-term survival –21. AccoWith regards to the impact of follow-up duration on the difference in aortic remodeling, the median follow-up duration for TEVAR and open repair were 37.5 and 40.3 months, respectively. All patients from both treatment groups had complete resolution of the IMH in the AA, and resolution all occurred within 1 year after TEVAR. However, TEVAR patients were at risk stent graft induced new entry tear and progression of intrinsic descending pathology. Studies have also shown a 10–40% risk of late reoperation for patients undergoing open repair , 34. TheOur findings were consistent with previous studies , suggestOur study was a single center retrospective study with a relatively small case number and non-consecutive case enrollment. The study cohort was heterogeneous in nature, which might have impact on data analysis and interpretation. Though general consensus on the indication for TEVAR or surgical aortic repair were in place, the ultimate decision remained at the discretion of the operating surgeon, after discussing the potential pros and cons with the patient. This might result in selection bias. The presence of selection bias was suggested by the following: (1) 72.7% of those who underwent open repair were operated within 24 h, compared to only 46.2% for the TEVAR cohort (2) More patients in the open repair group had severe aortic insufficiency and aneurysmal dilatation of the aorta prior to intervention (3) 72.7% of those who received open repair had an IMH thickness >11mm & ascending aortic diameter >50mm, in contrast to only 23.1% in the TEVAR group.Currently, the standard treatment for type A IMH based on existing guidelines is still open aortic repair. Though TEVAR has been reported by literatures with promising results in the treatment of retrograde type A IMH, it has not been regarded as a standard treatment modality for this particular patient group and the optimal treatment remains controversial among open surgical grafting, TEVAR or medical treatment. Therefore, a validated treatment algorithm to determine which patient should undergo TEVAR is currently lacking and it was one of the reason why this study was conducted. Currently, an ascending aortic IMH thickness > 11mm and a maximum ascending aortic diameter >50mm are in preference to open aortic repair, though the final decision may be greatly affected by factors including patient and physician preference. Further prospective studies with a larger cohort and longer follow-up are necessary in order to draw a firm conclusion on the indication and optimal treatment modality for retrograde type A IMH.This was the first study presenting outcome data on both TEVAR and open aortic repair in patients with retrograde type A IMH. In this study, TEVAR and open aortic repair were both effective treatment for retrograde type A IMH. TEVAR promoted remodeling of the AA with complete resolution of the IMH, whereas open aortic repair replaced the diseased AA entirely. From our results, TEVAR might also have the potential advantage of lower post-operative morbidity and better remodeling of the DTA. This study suggested that TEVAR might be a safe, effective alternative treatment modality in selected patients with retrograde type A IMH.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants this study was approved by the institutional review board of National Taiwan University Hospital (NTUH–202101046RINA) and the board waived the need for informed consent. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.KY: data curation and writing—original draft. N-HC: data curation, and review and editing. H-YY: data curation. Y-SC: writing—review and editing. S-SW: conceptualization and supervision. I-HW: conceptualization, methodology, writing—review and editing, validation, and supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.