Epidemiology and genetic diversity of linezolid resistant enterococci clinical isolates in Belgium from 2013 to 2021.

OBJECTIVES: Linezolid resistant opportunistic human pathogens Enterococcus faecalis and Enterococcus faecium are an emerging health threat as limited therapeutic options remain. The aim was to investigate the epidemiology, resistance mechanisms and genetic diversity of Belgian linezolid resistant enterococci (LRE) isolated between 2013 and 2021 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 (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 (STs) in linezolid resistant E. faecalis strains, while ST203 (n=5/15 typed strains, 33%) was the most frequently detected STs in linezolid resistant E. faecium strains. CONCLUSIONS: E. faecalis isolates harbouring optrA were the predominant LRE in Belgium with ST480 as the most prominent MLST. Linezolid resistance in E. faecium could be attributed to either chromosomal mutations or transferable resistance determinants.

Lack of functional TCR-epitope interaction is associated with herpes zoster through reduced downstream T cell activation.

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.

Unravelling the immune signature of herpes zoster: Insights into pathophysiology and the HLA risk profile.

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

Epidemiology and molecular typing of multidrug-resistant bacteria in tertiary hospitals and nursing homes in Flanders, Belgium.

This study aimed to map MDRO carriage and potential transmission within and between three Flemish tertiary care hospitals and their neighbouring nursing homes. A cross-sectional MDRO prevalence survey was organized between October 2017 and February 2019. Perianal swabs were cultured for detection of MDRO. Determination of clonal relatedness based on wgMLST allelic profiles was performed. The prevalence of MDRO in Belgian hospitals and NHs is on the rise, compared to previous studies, and transmission in and between institutions is observed. These results re-emphasize the need for a healthcare network-wide infection prevention strategy in which WGS of MDRO strains can be supportive.

Genital Infection Caused by Salmonella enterica Serovar Hvittingfoss: A Case Report.

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.

Epidemiology and molecular typing of multidrug-resistant bacteria in day care centres in Flanders, Belgium.

The global prevalence and spread of multidrug-resistant organisms (MDROs) represent an emerging public health threat. Day care centre (DCC) attendance is a risk factor for MDRO carriage in children and their environment. This study aimed to map the epidemiology of carriage and potential transmission of these organisms within 18 Flemish DDCs (Belgium). An MDRO prevalence survey was organised between November 2018 and February 2019 among children attending the centres. Selective chromogenic culture media were used for the detection of extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E), carbapenemase-producing Enterobacterales (CPE), and vancomycin-resistant Enterococci (VRE) in faecal swabs obtained from diapers or jars (n = 448). All isolated MDROs were subjected to resistance gene sequencing. A total of 71 of 448 samples (15.8%) yielded isolates of ESBL-E with a predominance of Escherichia coli (92.2% of ESBL-E) and ESBL resistance gene blaCTX-M-15 (50.7% of ESBL coding genes in E. coli). ESBL-E prevalence varied between DCCs, ranging from 0 to 50%. Transmission, based on the clonal relatedness of ESBL-E strains, was observed. CPE was identified in only one child carrying an E. coli with an OXA-244 gene. VRE was absent from all samples. The observed prevalence of ESBL-E in Flemish DCCs is high compared with previous studies, and our findings re-emphasise the need for rigorous hygiene measures within such centres to control the further spread of MDROs in the community.

Preanalytical variables influencing the interpretation and reporting of biological tests on blood samples of living and deceased donors for human body materials.

With the present paper, the Working Group on Cells, Tissues and Organs and other experts of the Superior Health Council of Belgium aimed to provide stakeholders in material of human origin with advice on critical aspects of serological and nucleic acid test (NAT) testing, to improve virological safety of cell- and tissue and organ donation. The current paper focusses on a number of preanalytical variables which can be critical for any medical biology examination: (1) sampling related variables (type of samples, collection of the samples, volume of the sample, choice of specific tubes, identification of tubes), (2) variables related to transport, storage and processing of blood samples (transport, centrifugation and haemolysis, storage before and after centrifugation, use of serum versus plasma), (3) variables related to dilution (haemodilution, pooling of samples), and (4) test dependent variables (available tests and validation). Depending on the type of donor (deceased donor (heart-beating or non-heart beating) versus living donor (allogeneic, related, autologous), and the type of donated human material (cells, tissue or organs) additional factors can play a role: pre- and post-mortem sampling, conditions of sampling (e.g. morgue), haemodilution, possibility of retesting.

Audit of a computerized version of the Manchester triage system and a SIRS-based system for the detection of sepsis at triage in the emergency department.

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).

Follow-up blood cultures in Staphylococcus aureus bacteremia: a probability-based optimization.

Staphylococcus aureus bacteremia (SAB) is a relevant finding which prompts a thorough diagnostic work-up. Follow-up blood cultures (BC) are essential in this work-up. We investigate the probability of detecting an ongoing bacteremia after initiation of active therapy according to the number of BC taken at key time points. A retrospective analysis of all patients with SAB in a 6-year period was performed. Total number of BCs taken and the positivity was registered for each day after start of therapy. A positivity-rate was corrected using a logistic mixed effects model. Observed detection frequencies were applied to calculate detection probabilities using binomial distributions. Three hundred and seventeen cases were withheld for analysis. A BC bottle positivity rate of 66.7% was found 1 day after initiation of active therapy, which decreased to 48.5% on day 4. When using 1 set of FU-BC, 73.4% of persisting SABs are detected. To maintain a probability of detection of ≥ 90%, 2 BC sets should be taken on day 2 and day 4 after start of therapy. In 10 of 109 patients with positive FU-BC, skip phenomena were registered, with a significant higher proportion in patients with < 4 BC bottles taken (14%) than when ≥ 4 BC bottles were taken (4.1%). We recommend taking 2 BC sets on days 2 and 4 after start of therapy in order to detect ≥ 90% of persisting SABs, limiting skip phenomena and blood volume required. We strongly advice against taking a single BC set as follow-up for SAB.

Evaluation of Saliva as a Matrix for RT-PCR Analysis and Two Rapid Antigen Tests for the Detection of SARS-CoV-2.

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.

Safety and Efficacy of Antibiotic De-escalation and Discontinuation in High-Risk Hematological Patients With Febrile Neutropenia: A Single-Center Experience.

BACKGROUND: There is currently no consensus on optimal duration of antibiotic treatment in febrile neutropenia. We report on the clinical impact of implementation of antibiotic de-escalation and discontinuation strategies based on the Fourth European Conference on Infections in Leukaemia (ECIL-4) recommendations in high-risk hematological patients. METHODS: We studied 446 admissions after introduction of an ECIL-4-based protocol (hereafter "ECIL-4 group") in comparison to a historic cohort of 512 admissions. Primary clinical endpoints were the incidence of infectious complications including septic shock, infection-related intensive care unit (ICU) admission, and overall mortality. Secondary endpoints included the incidence of recurrent fever, bacteremia, and antibiotic consumption. RESULTS: Bacteremia occurred more frequently in the ECIL-4 group (46.9% [209/446] vs 30.5% [156/512]; P < .001), without an associated increase in septic shock (4.7% [21/446] vs 4.5% [23/512]; P = .878) or infection-related ICU admission (4.9% [22/446] vs 4.1% [21/512]; P = .424). Overall mortality was significantly lower in the ECIL-4 group (0.7% [3/446] vs 2.7% [14/512]; P = .016), resulting mainly from a decrease in infection-related mortality (0.4% [2/446] vs 1.8% [9/512]; P = .058). Antibiotic consumption was significantly reduced by a median of 2 days on antibiotic therapy (12 vs 14; P = .001) and 7 daily antibiotic doses (17 vs 24; P < .001) per admission period. CONCLUSIONS: Our results support implementation of ECIL-4 recommendations to be both safe and effective based on real-world data in a large high-risk patient population. We found no increase in infectious complications and total antibiotic exposure was significantly reduced.

Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination.

Antigen recognition through the T cell receptor (TCR) αß 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 TCRß 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 TCRß 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.

Immune cells called CD4 T cells help the body build immunity to infections caused by bacteria and viruses, or after vaccination. Receptor proteins on the outside of the cells recognize pathogens, foreign molecules called antigens, or vaccine antigens. Vaccine antigens are usually inactivated bacteria or viruses, or fragments of these pathogens. After recognizing an antigen, CD4 T cells develop into memory CD4 T cells ready to defend against future infections with the pathogen. People who have never been exposed to a pathogen, or have never been vaccinated against it, may nevertheless have preexisting memory cells ready to defend against it. This happens because CD4 T cells can recognize multiple targets, which enables the immune system to be ready to defend against both new and familiar pathogens. Elias, Meysman, Bartholomeus et al. wanted to find out whether having preexisting memory CD4 T cells confers an advantage for vaccine-induced immunity. Thirty-four people who were never exposed to hepatitis B or vaccinated against it participated in the study. These individuals provided blood samples before vaccination, with 2 doses of the hepatitis B vaccine, and at 3 time points afterward. Using next generation immune sequencing and machine learning techniques, Elias et al. analyzed the individuals' memory CD4 T cells before and after vaccination. The experiments showed that preexisting memory CD4 T cells may determine vaccination outcomes, and people with more preexisting memory cells develop quicker and stronger immunity after vaccination against hepatitis B. This information may help scientists to better understand how people develop immunity to pathogens. It may guide them develop better vaccines or predict who will develop immunity after vaccination.

Monitoring the SARS-CoV-2 pandemic: screening algorithm with single nucleotide polymorphism detection for the rapid identification of established and emerging variants.

OBJECTIVES: To evaluate a testing algorithm for the rapid identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that includes the use of PCR-based targeted single nucleotide polymorphism (SNP) detection assays preceded by a multiplex PCR sensitive to S-Gene Target Failure (SGTF). METHODS: PCR SNP assays targeting SARS-CoV-2 S-gene mutations ΔH69-V70, L452R, E484K, N501Y, H655Y and P681R using melting curve analysis were performed on 567 samples in which SARS-CoV-2 viral RNA was detected by a multiplex PCR. Viral whole-genome sequencing (WGS) was performed to confirm the presence of SNPs and to identify the Pangolin lineage. Additionally, 1133 SARS-CoV-2 positive samples with SGTF were further assessed by WGS to determine the presence of ΔH69-V70. RESULTS: The N501Y-specific assay (n = 567) had an overall percentage agreement (OPA) of 98.5%. The ΔH69-V70-specific (n = 178) and E484K-specific (n = 401) assays had OPA of 96.6% and 99.7%, respectively. Assessment of H655Y (n = 139) yielded a 100.0% concordance when applied in the proposed algorithm. The L452R-specific (n = 67) and P681R-specific (n = 62) assays had an OPA of 98.2% and 98.1%, respectively. The proposed algorithm identified six variants of concern/interest (VOC/VOI)-Alpha (n = 149), Beta (n = 65), Gamma (n = 86), Delta (n = 49), Eta (n = 6), Kappa (n = 6)-and 205 non-VOC/VOI strains-including the variants under monitoring B.1.214.2 (n = 43) and B.1.1.318 (n = 18) and Epsilon (n = 1). An excellent concordance was observed for the identification of all SARS-CoV-2 lineages evaluated. CONCLUSIONS: We present a flexible testing algorithm for the rapid detection of current and emerging SARS-CoV-2 VOC/VOIs, which can be easily adapted based on the local endemicity of specific variants.

Laboratory analysis of two Delta SARS-CoV-2 variant outbreaks in the Port of Antwerp.

INTRODUCTION: The B.1.617.2 SARS-CoV-2 or Delta variant, first detected in India, has shown a rapid global spread due to its high transmissibility and now represents more than 99% of the currently circulating variants in Europe. METHODS AND RESULT: In May 2021, two ships that had recently arrived in the Port of Antwerp reported crew members with COVID-like symptoms. SARS-CoV-2 RNA was detected in nasopharyngeal swabs in 30 out of 45 skippers and the B.1.617.2 variant was identified via whole genome sequencing. Crew members were isolated or quarantined and repeatedly tested to assess the evolution of their SARS-CoV-2 viral load based on the cycle threshold (CT) values of the PCR reaction. Viral cultures were also taken at day 7 to detect viable virus and were compared with the subjects CT value at that moment. The shipper's clinical condition was closely observed using a digital home monitoring tool. Eleven crew members (37%) required hospitalization, with CT values of SARS-CoV-2 RNA being a good predictive factor for the hospitalization need. Furthermore, a clear correlation between CT values and positive viral culture was observed, hinting infectiousness even longer than 10 days after the intitial positive PCR test. CONCLUSION: Our study of 2 Delta variant clusters shows that the initial CT value is a good predictor for hospitalization need and suggests that patients infected with this variant may remain infectious for a longer time period.

Serological response in health care workers after a single dose of SARS-CoV-2 vaccine using six automated SARS-CoV-2 antibody assays.

Spike (S)- and nucleocapsid (N)-specific serological assay responses were determined before and/or after first dose SARS-CoV-2 vaccination in 22 individuals. S-specific assays quantified antibodies after vaccination with significant higher levels in participants with a previous infection. Be cautious combining N-/S-specific assay results, potentially differentiating post-infection/vaccination immunization as assay-specific N-antibody waning was observed.

A Pseudomonas monteilii unilateral inguinofemoral granulomatous lymphadenitis in a two-year-old girl. A case report.

Introduction: Pseudomonas monteilii is an environmental contaminant and is considered as an emerging human pathogen. We report the case of a Pseudomonas monteilii granulomatous lymphadenitis in a two-year-old girl.Clinical Presentation and Intervention: A two-year-old, previously healthy, Caucasian girl developed a unilateral inguinofemoral granulomatous lymphadenitis with Pseudomonas monteilii. The protracted course, the violaceous discoloration of the overlying skin, the mild tenderness without constitutional signs, the reactive tuberculin skin test with a negative interferon gamma release assay (IGRA) and the negative Bartonella henselae serology ranked non-tuberculous mycobacterial lymphadenitis high in our differential diagnosis. The ultrasonography showed signs of abcedation. We decided for surgical excision of the nodes. A P.monteilii granulomatous lymphadenitis was revealed. Treatment with an oral course of 2 weeks ciprofloxacin was prescribed. The course after treatment was uneventful and after one year of follow-up, the child is still doing well.Conclusions: Unusual clinical presentation should raise suspicion of uncommon pathogens and uncommon pathogens should raise suspicion of an underlying problem such as immunodeficiency, which was not the case in our patient.

Symptomatic Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection of a Healthcare Worker in a Belgian Nosocomial Outbreak Despite Primary Neutralizing Antibody Response.

BACKGROUND: It is currently unclear whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection will remain a rare event, only occurring in individuals who fail to mount an effective immune response, or whether it will occur more frequently when humoral immunity wanes following primary infection. METHODS: A case of reinfection was observed in a Belgian nosocomial outbreak involving 3 patients and 2 healthcare workers. To distinguish reinfection from persistent infection and detect potential transmission clusters, whole genome sequencing was performed on nasopharyngeal swabs of all individuals including the reinfection case's first episode. Immunoglobulin A, immunoglobulin M, and immunoglobulin G (IgG) and neutralizing antibody responses were quantified in serum of all individuals, and viral infectiousness was measured in the swabs of the reinfection case. RESULTS: Reinfection was confirmed in a young, immunocompetent healthcare worker as viral genomes derived from the first and second episode belonged to different SARS-CoV-2 clades. The symptomatic reinfection occurred after an interval of 185 days, despite the development of an effective humoral immune response following symptomatic primary infection. The second episode, however, was milder and characterized by a fast rise in serum IgG and neutralizing antibodies. Although contact tracing and viral culture remained inconclusive, the healthcare worker formed a transmission cluster with 3 patients and showed evidence of virus replication but not of neutralizing antibodies in her nasopharyngeal swabs. CONCLUSIONS: If this case is representative of most patients with coronavirus disease 2019, long-lived protective immunity against SARS-CoV-2 after primary infection might not be likely.

Evaluating SARS-CoV-2 spike and nucleocapsid proteins as targets for antibody detection in severe and mild COVID-19 cases using a Luminex bead-based assay.

Large-scale serosurveillance of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) will only be possible if serological tests are sufficiently reliable, rapid and affordable. Many assays are either labour-intensive and require specialised facilities (e.g. virus neutralization assays), or are expensive with suboptimal specificity (e.g. commercial ELISAs and RDTs). Bead-based assays offer a cost-effective alternative and allow for multiplexing to test for antibodies against multiple antigens and against other pathogens. Here, we compare the performance of spike (S) and nucleocapsid (NP) antigens for the detection of SARS-CoV-2 specific IgG, IgM and IgA antibodies in a panel of sera that includes recent (up to six weeks after symptom onset, severe n = 44; and mild cases n = 52) and old infections (five months after symptom onset, mild n = 104), using a Luminex-bead based assay and comparison to a virus neutralization test. While we show that neutralizing antibody levels are significantly lower in mild than in severe cases, we demonstrate that a combination of the recombinant nucleocapsid protein (NP) and receptor-binding domain (RBD) results in highly specific (99 %) IgG antibody detection five months after infection in 96 % of cases. Although most severe Covid-19 cases developed a clear IgM and IgA response, titers fell below the detection threshold in more than 20 % of mild cases in our bead-based assay. In conclusion, our data supports the use of RBD and NP for the development of SARS-CoV-2 serological IgG bead-based assays.

Fatal lymphocytic cardiac damage in coronavirus disease 2019 (COVID-19): autopsy reveals a ferroptosis signature.

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.

Transcriptomic profiling of different responder types in adults after a Priorix® vaccination.

Thanks to the recommendation of a combined Measles/Mumps/Rubella (MMR) vaccine, like Priorix®, these childhood diseases are less common now. This is beneficial to limit the spread of these diseases and work towards their elimination. However, the measles, mumps and rubella antibody titers show a large variability in short- and long-term immunity. The recent outbreaks worldwide of measles and mumps and previous studies, which mostly focused on only one of the three virus responses, illustrate that there is a clear need for better understanding the immune responses after vaccination. Our healthy cohort was already primed with the MMR antigens in their childhood. In this study, the adult volunteers received one Priorix® vaccine dose at day 0. First, we defined 4 different groups of responders, based on their antibody titers' evolution over 4 time points (Day 0, 21, 150 and 365). This showed a high variability within and between individuals. Second, we determined transcriptome profiles using 3'mRNA sequencing at day 0, 3 and 7. Using two analytical approaches, "one response group per time point" and "a time comparison per response group", we correlated the short-term gene expression profiles to the different response groups. In general, the list of differentially expressed genes is limited, however, most of them are clearly immune-related and upregulated at day 3 and 7, compared to the baseline day 0. Depending on the specific response group there are overlapping signatures for two of the three viruses. Antibody titers and transcriptomics data showed that an additional Priorix vaccination does not facilitate an equal immune response against the 3 viruses or among different vaccine recipients.