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Objectives: Linezolid-resistant opportunistic human pathogens Enterococcus faecalis and Enterococcus faecium are emerging health threats as limited therapeutic options remain. The aim of this study was to investigate the epidemiology, resistance mechanisms, and genetic diversity of linezolid-resistant enterococci (LRE) isolated between 2013 and 2021 and received at the Belgian National Reference Centre (NRC) for Enterococci. Methods: Linezolid susceptibility testing was performed upon request on 2458 submitted enterococci strains. Whole-genome sequencing was performed on all LRE strains. Results: Seventy-eight LRE human isolates, of which 63 (81%) E. faecalis and 15 (19%) E. faecium strains, were submitted to the Belgian NRC for Enterococci. Of the linezolid-resistant E. faecalis strains, 97% harboured the optrA gene (56% wild-type pE349) and 3% the poxtA gene. Of the linezolid-resistant E. faecium strains, 54% harboured the G2576T point mutation in the V domain of the 23S rRNA genes, 23% the poxtA, and 23% the optrA gene. Furthermore, two E. faecium strains were identified with a combination of two resistance mechanisms ([i] optrA and poxtA, and [ii] cfr(B) and G2576T point mutation, respectively). Vancomycin resistance was observed in 15% (n = 12) of the LRE. ST480 (n = 42/63 typed strains, 67%) was the most frequently detected sequence type (ST) in linezolid-resistant E. faecalis strains, while ST203 (n = 5/15 typed strains, 33%) was the most frequently detected ST in linezolid-resistant E. faecium strains. Conclusions: E. faecalis isolates harbouring optrA were the predominant LRE in Belgium, with ST480 as the most prominent multilocus sequence typing. Linezolid resistance in E. faecium could be attributed to either chromosomal mutations or transferable resistance determinants.

Summary: The role of T cell receptor (TCR) diversity in infectious disease susceptibility is not well understood. We use a systems immunology approach on three cohorts of herpes zoster (HZ) patients and controls to investigate whether TCR diversity against varicella-zoster virus (VZV) influences the risk of HZ. We show that CD4+ T cell TCR diversity against VZV glycoprotein E (gE) and immediate early 63 protein (IE63) after 1-week culture is more restricted in HZ patients. Single-cell RNA and TCR sequencing of VZV-specific T cells shows that T cell activation pathways are significantly decreased after stimulation with VZV peptides in convalescent HZ patients. TCR clustering indicates that TCRs from HZ patients co-cluster more often together than TCRs from controls. Collectively, our results suggest that not only lower VZV-specific TCR diversity but also reduced functional TCR affinity for VZV-specific proteins in HZ patients leads to lower T cell activation and consequently affects the susceptibility for viral reactivation.

Abstract Background and importance Different triage systems can be used to screen for sepsis and are often incorporated into local electronic health records. Often the design and interface of these digitalizations are not audited, possibly leading to deleterious effects on screening test performance. Objective To audit a digital version of the MTS for detection of sepsis during triage in the ED. Design A single-center retrospective study Settings and participants Patients (n=29766) presenting to an ED of a tertiary-care center who received formal triage were included. Outcome measures and analysis Calculated performance measures included sensitivity, specificity, likelihood ratios, and AUC for the detection of sepsis. Errors in the application of the specific sepsis discriminator of the MTS were recorded. Main results A total of 189 (0.7%) subjects met the Sepsis-3 criteria, with 47 cases meeting the criteria for septic shock. The MTS had a low sensitivity of 47.6% (95% CI 40.3 to 55.0) for allocating sepsis patients to the correct triage category. However, specificity was high at 99.4% (95% CI 99.3 to 99.5).

Liver-resident NK (lrNK) cells have been studied in humans as well as in mice. Unfortunately, important differences have been observed between murine and human lrNK cells, complicating the extrapolation of data obtained in mice to man. We previously described two NK cell subsets in the porcine liver: A CD8αhigh subset, with a phenotype much like conventional CD8αhigh NK cells found in the peripheral blood, and a specific liver-resident CD8αdim subset which phenotypically strongly resembles human lrNK cells. These data suggest that the pig might be an attractive model for studying lrNK cell biology. In the current study, we used RNA-seq to compare the transcriptome of three porcine NK cell populations: Conventional CD8αhigh NK cells from peripheral blood (cNK cells), CD8αhigh NK cells isolated from the liver, and the liver-specific CD8αdim NK cells. We found that highly expressed transcripts in the CD8αdim lrNK cell population mainly include genes associated with the (adaptive) immune response, whereas transcripts associated with cell migration and extravasation are much less expressed in this subset compared to cNK cells. Overall, our data indicate that CD8αdim lrNK cells show an immature and anti-inflammatory phenotype. Interestingly, we also observed that the CD8αhigh NK cell population that is present in the liver appears to represent a population with an intermediate phenotype. Indeed, while the transcriptome of these cells largely overlaps with that of cNK cells, they also express transcripts associated with liver residency, in particular CXCR6. The current, in-depth characterization of the transcriptome of porcine liver NK cell populations provides a basis to use the pig model for research into liver-resident NK cells.

Summary: The mechanisms by which viruses regulate host mRNAs during infection are still poorly understood. Several host transcripts that encode proteins that contribute to the anti-viral response contain the N6-methyladenosine nucleotide (m6A). In this study, we investigated if and how viruses from different (sub) families specifically affect m6A-containing host transcripts. Systematic analysis of host transcriptomes after infection with diverse types of viruses showed that m6A-methylated transcripts are selectively downregulated during infection with Sendai virus, African swine fever virus and the alphaherpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV). Focusing on PRV and HSV-1, we found that downregulation of m6A-methylated transcripts depends on the YTHDF family of m6A-binding proteins, and correlates with localization of these proteins to enlarged P-bodies. Knockdown of YTHDF proteins in primary cells reduced PRV protein expression and increased expression of antiviral interferon-stimulated genes, suggesting that virus-induced depletion of host m6A-containing transcripts constitutes an immune evasion strategy.

Background: Nontyphoidal Salmonella serovars predominantly cause gastrointestinal infections. However, other clinical presentations, including urogenital infections, have been reported, although they are rather rare. Case presentation: This case is about a 33-year-old woman diagnosed with Salmonella enterica serovar Hvittingfoss (S. Hvittingfoss) bacteremia and endometritis six days post uterine aspiration in the context of a missed abortion. She had traveled to Indonesia two weeks prior to the positive blood and cervical culture. She never developed gastrointestinal symptoms but was found to carry S. Hvittingfoss in her stool sample. The patient was successfully treated with a seven-day course of iv ciprofloxacin. Conclusions: S. Hvittingfoss is a rare serovar that has caused a few outbreaks of foodborne infections in Asia, the United States, and Australia. To the best of our knowledge, this is the first reported case of Salmonella urogenital infection caused by this serovar. Salmonella as a cause of urogenital infections is rare but not uncommon. Therefore, it should be considered in identifying members of the Enterobacterales among urogenital flora in cases of severe urogenital infections, especially when other cultures remain negative.

Abstract Small intestinal organoids, or enteroids, represent a valuable model to study host–pathogen interactions at the intestinal epithelial surface. Much research has been done on murine and human enteroids, however only a handful studies evaluated the development of enteroids in other species. Porcine enteroid cultures have been described, but little is known about their functional responses to specific pathogens or their associated virulence factors. Here, we report that porcine enteroids respond in a similar manner as in vivo gut tissues to enterotoxins derived from enterotoxigenic Escherichia coli, an enteric pathogen causing postweaning diarrhoea in piglets. Upon enterotoxin stimulation, these enteroids not only display a dysregulated electrolyte and water balance as shown by their swelling, but also secrete inflammation markers. Porcine enteroids grown as a 2D-monolayer supported the adhesion of an F4+ ETEC strain. Hence, these enteroids closely mimic in vivo intestinal epithelial responses to gut pathogens and are a promising model to study host–pathogen interactions in the pig gut. Insights obtained with this model might accelerate the design of veterinary therapeutics aimed at improving gut health.

The aim of this research was to study roles, strengths and challenges of robot-mediated interventions using robot KASPAR for children with autism spectrum disorder (ASD). Twelve focus group sessions were organized in which 70 ASD care and/or education professionals participated. Six roles for KASPAR were identified: provoker, reinforcer, trainer, mediator, prompter, and diagnostic information provider. Strengths of KASPAR are related to personalisation possibilities, its playfulness, the action-reaction principle, its neutral expression, consistent and repetitive application of actions, possibilities to vary behaviour in a controlled manner and having an extra hand. Challenges of working with KASPAR were: limited reaction possibilities, possibility of children being scared of KASPAR, difficulties with generalisation or transfer and finally potential dependence on KASPAR.

The varicella-zoster virus (VZV) infects >95% of the population. VZV reactivation causes herpes zoster (HZ), known as shingles, primarily affecting the elderly and individuals who are immunocompromised. However, HZ can occur in otherwise healthy individuals. We analyzed the immune signature and risk profile in patients with HZ using a genome-wide association study across different UK Biobank HZ cohorts. Additionally, we conducted one of the largest HZ human leukocyte antigen association studies to date, coupled with transcriptomic analysis of pathways underlying HZ susceptibility. Our findings highlight the significance of the major histocompatibility complex locus for HZ development, identifying 5 protective and 4 risk human leukocyte antigen alleles. This demonstrates that HZ susceptibility is largely governed by variations in the major histocompatibility complex. Furthermore, functional analyses revealed the upregulation of type I interferon and adaptive immune responses. These findings provide fresh molecular insights into the pathophysiology and activation of innate and adaptive immune responses triggered by symptomatic VZV reactivation. [ABSTRACT FROM AUTHOR]

Abstract Aims Cardiovascular complications, including myocarditis, are observed in coronavirus disease 2019 (COVID‐19). Major cardiac involvement is a potentially lethal feature in severe cases. We sought to describe the underlying pathophysiological mechanism in COVID‐19 lethal cardiogenic shock. Methods and results We report on a 48‐year‐old male COVID‐19 patient with cardiogenic shock; despite extracorporeal life support, dialysis, and massive pharmacological support, this rescue therapy was not successful. Severe acute respiratory syndrome coronavirus 2 RNA was detected at autopsy in the lungs and myocardium. Histopathological examination revealed diffuse alveolar damage, proliferation of type II pneumocytes, lymphocytes in the lung interstitium, and pulmonary microemboli. Moreover, patchy muscular, sometimes perivascular, interstitial mononuclear inflammatory infiltrates, dominated by lymphocytes, were seen in the cardiac tissue. The lymphocytes ‘interlocked’ the myocytes, resulting in myocyte degeneration and necrosis. Predominantly, T‐cell lymphocytes with a CD4:CD8 ratio of 1.7 infiltrated the interstitial myocardium, reflecting true myocarditis. The myocardial tissue was examined for markers of ferroptosis, an iron‐catalysed form of regulated cell death that occurs through excessive peroxidation of polyunsaturated fatty acids. Immunohistochemical staining with E06, a monoclonal antibody binding to oxidized phosphatidylcholine (reflecting lipid peroxidation during ferroptosis), was positive in morphologically degenerating and necrotic cardiomyocytes adjacent to the infiltrate of lymphocytes, near arteries, in the epicardium and myocardium. A similar ferroptosis signature was present in the myocardium of a COVID‐19 subject without myocarditis. In a case of sudden death due to viral myocarditis of unknown aetiology, however, immunohistochemical staining with E06 was negative. The renal proximal tubuli stained positively for E06 and also hydroxynonenal (4‐HNE), a reactive breakdown product of the lipid peroxides that execute ferroptosis. In the case of myocarditis of other aetiology, the renal tissue displayed no positivity for E06 or 4‐HNE. Conclusions The findings in this case are unique as this is the first report on accumulated oxidized phospholipids (or their breakdown products) in myocardial and renal tissue in COVID‐19. This highlights ferroptosis, proposed to detrimentally contribute to some forms of ischaemia–reperfusion injury, as a detrimental factor in COVID‐19 cardiac damage and multiple organ failure.

BackgroundEuropean surgeons were the first worldwide to use robotic techniques in cardiac surgery and major steps in procedure development were taken in Europe. After a hype in the early 2000s case numbers decreased but due to technological improvements renewed interest can be noted. We assessed the current activities and outcomes in robotically assisted cardiac surgery on the European continent.MethodsData were collected in an international anonymized registry of 26 European centers with a robotic cardiac surgery program.ResultsDuring a 4-year period (2016–2019), 2,563 procedures were carried out [30.0% female, 58.5 (15.4) years old, EuroSCORE II 1.56 (1.74)], including robotically assisted coronary bypass grafting (n = 1266, 49.4%), robotic mitral or tricuspid valve surgery (n = 945, 36.9%), isolated atrial septal defect closure (n = 225, 8.8%), left atrial myxoma resection (n = 54, 2.1%), and other procedures (n = 73, 2.8%). The number of procedures doubled during the study period (from n = 435 in 2016 to n = 923 in 2019). The mean cardiopulmonary bypass time in pump assisted cases was 148.6 (63.5) min and the myocardial ischemic time was 88.7 (46.1) min. Conversion to larger thoracic incisions was required in 56 cases (2.2%). Perioperative rates of revision for bleeding, stroke, and mortality were 56 (2.2%), 6 (0.2 %), and 27 (1.1%), respectively. Median postoperative hospital length of stay was 6.6 (6.6) days.ConclusionRobotic cardiac surgery case numbers in Europe are growing fast, including a large spectrum of procedures. Conversion rates are low and clinical outcomes are favorable, indicating safe conduct of these high-tech minimally invasive procedures.

Mucormycosis is a rare, emerging angioinvasive infection caused by ubiquitous filamentous fungi. In recent decades, an increase in cutaneous or post-traumatic mucormycosis has been reported. We describe two cases of post-traumatic wound infections with Mucor circinelloides, a mucor species only rarely reported as a cause of post-traumatic mucormycosis. Often considered lethal, management required a combination of medical and surgical therapies to achieve a favorable outcome in both cases.

Antigen recognition through the T cell receptor (TCR) αβ heterodimer is one of the primary determinants of the adaptive immune response. Vaccines activate naïve T cells with high specificity to expand and differentiate into memory T cells. However, antigen-specific memory CD4 T cells exist in unexposed antigen-naïve hosts. In this study, we use high-throughput sequencing of memory CD4 TCRβ repertoire and machine learning to show that individuals with preexisting vaccine-reactive memory CD4 T cell clonotypes elicited earlier and higher antibody titers and mounted a more robust CD4 T cell response to hepatitis B vaccine. In addition, integration of TCRβ sequence patterns into a hepatitis B epitope-specific annotation model can predict which individuals will have an early and more vigorous vaccine-elicited immunity. Thus, the presence of preexisting memory T cell clonotypes has a significant impact on immunity and can be used to predict immune responses to vaccination.

Abstract Background Meningitis can be caused by several viruses and bacteria. Identifying the causative pathogen as quickly as possible is crucial to initiate the most optimal therapy, as acute bacterial meningitis is associated with a significant morbidity and mortality. Bacterial meningitis requires antibiotics, as opposed to enteroviral meningitis, which only requires supportive therapy. Clinical presentation is usually not sufficient to differentiate between viral and bacterial meningitis, thereby necessitating cerebrospinal fluid (CSF) analysis by PCR and/or time-consuming bacterial cultures. However, collecting CSF in children is not always feasible and a rather invasive procedure. Methods In 12 Belgian hospitals, we obtained acute blood samples from children with signs of meningitis (49 viral and 7 bacterial cases) (aged between 3 months and 16 years). After pathogen confirmation on CSF, the patient was asked to give a convalescent sample after recovery. 3′ mRNA sequencing was performed to determine differentially expressed genes (DEGs) to create a host transcriptomic profile. Results Enteroviral meningitis cases displayed the largest upregulated fold change enrichment in type I interferon production, response and signaling pathways. Patients with bacterial meningitis showed a significant upregulation of genes related to macrophage and neutrophil activation. We found several significantly DEGs between enteroviral and bacterial meningitis. Random forest classification showed that we were able to differentiate enteroviral from bacterial meningitis with an AUC of 0.982 on held-out samples. Conclusions Enteroviral meningitis has an innate immunity signature with type 1 interferons as key players. Our classifier, based on blood host transcriptomic profiles of different meningitis cases, is a possible strong alternative for diagnosing enteroviral meningitis.

The use of saliva for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sparks debate due to presumed lower sensitivity and lack of standardization. Our aim was to evaluate the performance characteristics of (i) saliva collected by the ORAcollectTM device as a matrix for SARS-CoV-2 reverse-transcriptase polymerase chain reaction (RT-PCR), and (ii) 2 saliva rapid antigen tests (AgRDT). From 342 ambulatory individuals, both a nasopharyngeal swab and saliva sample via ORAcollectTM were obtained for a SARS-CoV-2 RT-PCR test. Furthermore, 54 and 123 additionally performed the V-ChekTM or WhistlingTM saliva AgRDT. In total, 35% of individuals screened positive for SARS-CoV-2 via nasopharyngeal swab. Saliva, as a matrix for the RT-PCR, had a specificity of 96.5% and a negative predictive value (NPV) of 91.3%. Interestingly, 6 out of 8 patients thought to be false positive in saliva re-tested positive by nasopharyngeal sampling after 2 to 9 days. Both V-ChekTM and WhistlingTM AgRDT had a lack of sensitivity, resulting in an NPV of 66.9 and 67.3%, respectively. Saliva proved to be a sensitive and specific matrix for SARS-CoV-2 detection by the RT-PCR. In this setting, saliva might have an earlier window of detection than the nasopharyngeal swab. By contrast, both AgRDT showed an unacceptably low sensitivity and NPV.

The aim of this study was to gain insight into how robots can be practically implemented into current education and therapy interventions for children with autism spectrum disorder (ASD). This qualitative study included focus groups and co-creation sessions. 73 Participants (professionals and adults with ASD) took part in 13 focus groups to elicit requirements for robot assisted interventions. Additionally, 22 participants (professionals, parents of children with ASD and adults with ASD) generated ideas for interventions using robot KASPAR in three co-creation sessions. This study resulted in: an overview of requirements concerning the robot, end-user, environment and practical implementation; a template to systematically describe robot interventions in general and for KASPAR in particular; and finally new interventions.