OBJECTIVES:Infection prevention and control (IPC) and antimicrobial stewardship (AMS) measures are critical to reducing transmission and infection by Clostridioides difficile (CDI) and other enteric pathogens. This study evaluated the impact of enhanced IPC and AMS on CDI and bloodstream infections (BSIs) caused by vancomycin-resistant enterococci (VRE) and third-generation cephalosporin-resistant Enterobacterales (3GCREB). METHODS:The study was conducted in five German university hospitals from January 2016 to July 2019. IPC and AMS interventions were sequentially enhanced in three departments with high-incidence CDI at baseline using a stepped-wedge cluster intervention approach. Main outcome measures were incidence densities of CDI and BSI caused by VRE and 3GCREB. An interrupted time series analysis was performed to assess the intervention effects during a normalized study period. RESULTS:Across 15 departments, >384,000 patient days were included. Incidence density of target infections was low (CDI, 0.77; VRE BSI, 0.07; and 3GCREB BSI, 0.09 per 1000 patient days). Pooled interrupted time series analysis results showed a significant reduction in CDI incidence density following the enhancement of AMS measures (AMS period regression slopes difference, -0.089; F[p] = 5.400 [0.037]). Regarding the incidence density of VRE/3GCREB BSI, no relevant changes could be observed (regression slopes difference, -0.19; F[p] = 0.667 [0.429]). A subgroup analysis focusing on haematological and oncological departments showed that AMS influenced prescription behaviour according to implemented AMS strategies, but not clinical outcomes. DISCUSSION:Combined with IPC enhanced short-term AMS measures led to a significant reduction in the incidence of CDI, whereas the incidence of BSI by VRE and 3GCREB remained unchanged in sites with well-established baseline IPC and AMS programmes and low incidence of hospital-associated infections.

Abstract Background Influenza is the most common vaccine-preventable infection among travelers, affecting approximately one percent of those travelling to subtropical and tropical destinations. Methods We analysed demographic, travel-related and clinical information from travelers diagnosed with influenza at our travel clinic between January 2015 and March 2020 and influenza-negative controls. Results We included 68 travelers diagnosed with influenza and 207 controls. In total, 22.1% of influenza patients (n = 15) were older than 60 years and/or had comorbidities for which annual influenza vaccination is recommended, but only one had received an influenza vaccine. Patients with respiratory and musculoskeletal symptoms who presented during the German influenza season had the highest risk proportion of positive tests (54%, n = 25/46). Overall, three (4.4%) influenza patients were hospitalised, two (2.9%) received antiviral treatment, and eight (11.8%) received antibiotic therapy. Conclusions Influenza occurs throughout the year in international travelers and can cause significant morbidity. Travelers with febrile illness should be tested for influenza, especially if they have respiratory or musculoskeletal symptoms, present during the local influenza season, or have travelled to South-East Asia. Influenza vaccination coverage among international travelers needs to be improved among high-risk individuals.

Purpose Hypertoxigenic Streptococcus pyogenes emm 1 lineage M1 UK has recently been associated with upsurges of invasive infections and scarlet fever in several countries, but whole-genome sequencing surveillance data of lineages circulating in Germany is lacking. In this study, we investigated recent iGAS isolates from our laboratory at a German tertiary care center for the presence of the M1 UK lineage. Methods Whole-genome sequencing was employed to characterize a collection of 47 consecutive non-copy isolates recovered from blood cultures (21) and tissue samples (26) in our laboratory between October 2022 and April 2023. Results M protein gene ( emm ) typing distinguished 14 different emm types, with emm 1 (17) being the dominant type. Single-nucleotide polymorphism (SNP) analysis confirmed the presence of all 27 SNPs characteristic for the M1 UK lineage in 14 of 17 emm 1 isolates. Conclusion This study has shown for the first time that M1 UK is present in Germany and might constitute a driving force in the observed surge of GAS infections. This observation mirrors developments in the UK and other countries and underscores the importance of WGS surveillance to understand the epidemiology of GAS.

Abstract Mortality of patients hospitalized with COVID-19 has remained high during the consecutive SARS-CoV-2 pandemic waves. Early discrimination of patients at high mortality risk is crucial for optimal patient care. Symmetric (SDMA) and asymmetric dimethylarginine (ADMA) have been proposed as possible biomarkers to improve risk prediction of COVID-19 patients. We measured SDMA, ADMA, and other L-arginine-related metabolites in 180 patients admitted with COVID-19 in four German university hospitals as compared to 127 healthy controls. Patients were treated according to accepted clinical guidelines and followed-up until death or hospital discharge. Classical inflammatory markers (leukocytes, CRP, PCT), renal function (eGFR), and clinical scores (SOFA) were taken from hospital records. In a small subgroup of 23 COVID-19 patients, sequential blood samples were available and analyzed for biomarker trends over time until 14 days after admission. Patients had significantly elevated SDMA, ADMA, and L-ornithine and lower L-citrulline concentrations than controls. Within COVID-19 patients, SDMA and ADMA were significantly higher in non-survivors (n = 41, 22.8%) than in survivors. In ROC analysis, the optimal cut-off to discriminate non-survivors from survivors was 0.579 µmol/L for SDMA and 0.599 µmol/L for ADMA (both p < 0.001). High SDMA and ADMA were associated with odds ratios for death of 11.45 (3.37–38.87) and 5.95 (2.63–13.45), respectively. Analysis of SDMA and ADMA allowed discrimination of a high-risk (mortality, 43.7%), medium-risk (15.1%), and low-risk group (3.6%); risk prediction was significantly improved over classical laboratory markers. We conclude that analysis of ADMA and SDMA after hospital admission significantly improves risk prediction in COVID-19.

We performed autopsies on persons in Germany who died from COVID-19 and observed higher nasopharyngeal SARS-CoV-2 viral loads for variants of concern (VOC) compared with non-VOC lineages. Pulmonary inflammation and damage appeared higher in non-VOC than VOC lineages until adjusted for vaccination status, suggesting COVID-19 vaccination may mitigate pulmonary damage.

BACKGROUND:Worldwide observations revealed increased frequencies of multi-resistant Enterobacterales and resistance genes in hospital wastewater compared to any other type of wastewater. Despite the description of clonal lineages possibly adapted to hospital wastewater, little is known about long term persistence as well as evolution of these lineages. METHODS:In this study, wastewater isolates of different Enterobacterales species from a tertiary care hospital were investigated with 2.5 years distance. Whole Genome Sequencing (WGS) and resistance gene identification were performed for E. coli, C. freundii, S. marcescens, K. pneumoniae, K. oxytoca, and E. cloacae isolates (n = 59), isolated in 2022 and compared with strains isolated from the same wastewater pipeline in 2019 (n = 240). RESULTS:Individual clonal lineages with highly related isolates could be identified in all species identified more than once in 2022 that appear to persist in the wastewater drainage. A common motif of all persistent clonal lineages was the carriage of mobile genetic elements encoding carbapenemase genes with hints for horizontal gene transfer in persistent clones in this environment observed over the 2.5-year period. Multiple plasmid replicons could be detected in both years. In 2022 isolates blaVIM-1 replaced blaOXA-48 as the most common carbapenemase gene compared to 2019. Interestingly, despite a similar abundance of carbapenemase genes (>80% of all isolates) at both time points genes encoding extended spectrum β-lactamases decreased over time. CONCLUSIONS:This data indicates that hospital wastewater continuously releases genes encoding carbapenemases to the urban wastewater system. The evolution of the resident clones as well as the reasons for the selection advantage in this specific ecological niche needs to be further investigated in the future.

BACKGROUND:In recent years, an increasing number of linezolid-resistant enterococci (LRE) was recognized at the German National Reference Centre (NRC) for Enterococci. National guidelines on infection prevention recommend screening for LRE in epidemiologically linked hospital settings without referring to a reliable and rapid diagnostic method. Since 2020, CHROMAgar™ provide a chromogenic linezolid screening agar, LIN-R, suitable to simultaneously screen for linezolid-resistant staphylococci and enterococci. OBJECTIVES:To assess the applicability of CHROMAgar™ LIN-R in clinical settings for detecting LRE directly from patient material and to infer prevalence rates of LRE amongst German hospital patients. METHODS:During the 3-month trial period, clinical samples were plated on CHROMAgar™ LIN-R. Antimicrobial susceptibility testing was performed using VITEK2 or disc diffusion. At the NRC, linezolid resistance was determined by broth microdilution, multiplex-PCR for cfr/optrA/poxtA and by a restriction-based assay for 23S rDNA mutations. RESULTS:The 12 participating study sites used 13 963 CHROMAgar™ LIN-R plates during the study period. Of 442 presumptive LRE, 192 were confirmed by phenotypic methods. Of these, 161 were received by the NRC and 121 (75%) were verified as LRE. Most of LR-E. faecium 53/81 (65%) exhibited a 23S rRNA gene mutation as the sole resistance-mediating mechanism, whereas optrA constituted the dominant resistance trait in LR-E. faecalis [39/40 (98%)]. Prevalence of LRE across sites was estimated as 1% (ranging 0.18%-3.7% between sites). CONCLUSIONS:CHROMAgar™ LIN-R represents a simple and efficient LRE screening tool in hospital settings. A high proportion of false-positive results demands validation of linezolid resistance by a reference method.

Despite therapeutic advances in the prevention and treatment of febrile neutropenia, acute leukemia (AL) patients still have considerable febrile neutropenia-related mortality. However, the diagnostic yield of flexible bronchoscopy (FB) and bronchoalveolar lavage (BAL) in acute leukemia patients is unclear. In this retrospective single-center study, we analyzed 88 BAL samples of patients with acute leukemia and pulmonary infiltrates in spite of treatment with broad-spectrum anti-infective agents. The aim was to investigate the impact of FB with BAL on detecting causative organisms, which would result in a change in treatment regimens. The median age was 59 years, and 86% had acute myeloid leukemia. In 47%, pathogens were detectable in BAL fluid (pathogen bacteria, viruses, and fungi in 2, 15, and 18%, respectively), with Aspergillus fumigatus detected most frequently. BAL-guided anti-infective therapy changes were performed in 15%. The detection of herpes simplex and influenza viruses were the main reasons for treatment changes. Despite broad-spectrum anti-infective treatment, in approximately half of all patients, pathogens could still be isolated in BAL samples. However, consecutive changes in anti-infective treatment were considerably less frequent, with most changes performed in patients with Herpes simplex virus and Influenza A detection. The need for FB with BAL in patients with AL receiving broad-spectrum empiric anti-infective treatment should therefore be weighed carefully.

BACKGROUND & AIMS:Detailed information on the immune response after second vaccination of cirrhotic patients and liver transplant (LT) recipients against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is largely missing. We aimed at comparing the vaccine-induced humoral and T-cell responses of these vulnerable patient groups. METHODS:In this prospective cohort study, anti-SARS-CoV-2 spike-protein titers were determined using the DiaSorin LIAISON (anti-S trimer) and Roche Elecsys (anti-S RBD) immunoassays in 194 patients (141 LT, 53 cirrhosis Child-Pugh A-C) and 56 healthy controls before and 10 to 84 days after second vaccination. The spike-specific T-cell response was assessed using an interferon-gamma release assay (EUROIMMUN). A logistic regression analysis was performed to identify predictors of low response. RESULTS:After the second vaccination, seroconversion was achieved in 63% of LT recipients and 100% of cirrhotic patients and controls using the anti-S trimer assay. Median anti-SARS-CoV-2 titers of responding LT recipients were lower compared with cirrhotic patients and controls (P < .001). Spike-specific T-cell response rates were 36.6%, 65.4%, and 100% in LT, cirrhosis, and controls, respectively. Altogether, 28% of LT recipients did neither develop a humoral nor a T-cell response after second vaccination. In LT recipients, significant predictors of absent or low humoral response were age >65 years (odds ratio [OR], 4.57; 95% confidence interval [CI], 1.48-14.05) and arterial hypertension (OR, 2.50; 95% CI, 1.10-5.68), whereas vaccination failure was less likely with calcineurin inhibitor monotherapy than with other immunosuppressive regimens (OR, 0.36; 95% CI, 0.13-0.99). CONCLUSION:Routine serological testing of the vaccination response and a third vaccination in patients with low or absent response seem advisable. These vulnerable cohorts need further research on the effects of heterologous vaccination and intermittent reduction of immunosuppression before booster vaccinations.

investigated of severe acute finding nasopharyngeal viral RNA stability in no time-dependent decrease. infectivity by virus iso lation to 35 hours postmortem.

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA loads in patient specimens may act as a clinical outcome predictor in critically ill patients with coronavirus disease 2019 (COVID-19). Methods We evaluated the predictive value of viral RNA loads and courses in the blood compared with the upper and lower respiratory tract loads of critically ill COVID-19 patients. Daily specimen collection and viral RNA quantification by reverse transcription quantitative polymerase chain reaction were performed in all consecutive 170 COVID-19 patients between March 2020 and February 2021 during the entire intensive care unit (ICU) stay (4145 samples analyzed). Patients were grouped according to their 90-day outcome as survivors (n=100) or nonsurvivors (n=70). Results In nonsurvivors, blood SARS-CoV-2 RNA loads were significantly higher at the time of admission to the ICU (P=.0009). Failure of blood RNA clearance was observed in 33/50 (66%) of the nonsurvivors compared with 12/64 (19%) survivors (P<.0001). As determined by multivariate analysis, taking sociodemographic and clinical parameters into account, blood SARS-CoV-2 RNA load represents a valid and independent predictor of outcome in critically ill COVID-19 patients (odds ratio [OR; log10], 0.23; 95% CI, 0.12–0.42; P<.0001), with a significantly higher effect for survival compared with respiratory tract SARS-CoV-2 RNA loads (OR [log10], 0.75; 95% CI, 0.66–0.85; P<.0001). Blood RNA loads exceeding 2.51×103 SARS-CoV-2 RNA copies/mL were found to indicate a 50% probability of death. Consistently, 29/33 (88%) nonsurvivors with failure of virus clearance exceeded this cutoff value constantly. Conclusions Blood SARS-CoV-2 load is an important independent outcome predictor and should be further evaluated for treatment allocation and patient monitoring.

Introduction. Laboratories worldwide are facing high demand for molecular testing during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, which might be further aggravated by the upcoming influenza season in the northern hemisphere. Gap Statement. Given that the symptoms of influenza are largely indistinguishable from those of coronavirus disease 2019 (COVID-19), both SARS-CoV-2 and the influenza viruses require concurrent testing by RT-PCR in patients presenting with symptoms of respiratory tract infection. Aim. We adapted and evaluated a laboratory-developed multiplex RT-PCR assay for simultaneous detection of SARS-CoV-2 (dual target), influenza A and influenza B (SC2/InflA/InflB-UCT) on a fully automated high-throughput system (cobas6800). Methodology. Analytical performance was assessed by serial dilution of quantified reference material and cell culture stocks in transport medium, including pretreatment for chemical inactivation. For clinical evaluation, residual portions of 164 predetermined patient samples containing SARS-CoV-2 ( n =52), influenza A ( n =43) or influenza B ( n =19), as well as a set of negative samples, were subjected to the novel multiplex assay. Results. The assay demonstrated comparable analytical performance to currently available commercial tests, with limits of detection of 94.9 cp ml −1 for SARS-CoV-2, 14.6 cp ml −1 for influenza A and 422.3 cp ml −1 for influenza B. Clinical evaluation showed excellent agreement with the comparator assays (sensitivity of 98.1, 97.7 and 100 % for Sars-CoV-2 and influenza A and B, respectively). Conclusion. The SC2/InflA/InflB-UCT allows for efficient high-throughput testing for all three pathogens and thus provides streamlined diagnostics while conserving resources during the influenza season.

1AbstractBackgroundLaboratories worldwide face high demands for molecular testing during the SARS-CoV-2 pandemic that might be further aggravated with the upcoming influenza season in the northern hemisphere. Considering that symptoms of influenza are largely undistinguishable from COVID-19, both SARS-CoV-2 and the Influenza viruses require concurrent testing by RT-PCR in patients presenting with symptoms of respiratory tract infection. In this study, we adapted and evaluated a laboratory developed multiplex RT-PCR assay for simultaneous detection of SARS-CoV-2 (dual-target), Influenza-A and Influenza-B (SC2/InflA/InflB-UCT) on a fully automated high-throughput system (cobas6800).MethodsAnalytical performance was assessed by serial dilution of quantified reference material and cell culture stocks in transport medium, including pre-treatment for chemical inactivation. For clinical evaluation, residual portions of 164 predetermined patient samples containing SARS-CoV-2 (n=52), Influenza-A (n=43) or Influenza-B (n=19), as well as a set of negative samples was subjected to the novel multiplex assay.ResultsThe assay demonstrated analytical performance comparable to currently available commercial tests, with limits of detection of 94.9 cp/ml for SARS-CoV-2, 14.6 cp/ml for Influenza-A and 422.3 cp/ml for Influenza-B. Clinical evaluation showed excellent agreement with the comparator assays (sensitivity 98.1%, 97.7% and 100% for Sars-CoV-2, Influenza-A and -B respectively).ConclusionThe SC2/InflA/InflB-UCT allows for efficient high-throughput testing for all three pathogens and thus provides streamlined diagnostics while conserving resources during the Influenza-season.HighlightsSimultaneous detection of highly pathogenic respiratory viruses Influenza-A/B and SARS-CoV-2Including a dual-target assay for SARS-CoV-2 detectionFull automation on the cobas6800 high-throughput platform

Increasing worldwide, prevalence of carbapenem-resistant gram-negative bacteria demands urgent a need for rapid detection and accurate identification of carbapenemases. The BD Phoenix CPO detect (PCD) assay possesses an in-built capacity for parallel susceptibility testing and detection of carbapenemases. Here, the ability of the assay to detect and classify carbapenemase production was tested in a collection of carbapenem-resistant Enterobacterales and non-fermentative gram-negative rods. The ability of the PCD assay to detect and classify carbapenemases was investigated in a collection of 194 clinical, carbapenem-resistant isolates ( Enterobacterales [ n = 65]; non-fermentative gram-negative rods [ n = 129]). AST results were compared to MICS determined by gradient diffusion to determine accuracy of the PCD assay. The accuracy of the PCD assay to detect carbapenemases was compared to the results of molecular isolate characterization using a LDT multiplex carbapenemase PCR assay. All 194 isolates classified as carbapenem-resistant by reference susceptibility testing were also classified correctly as CRO by the PCD assay. Performance analysis of the PCD assay to detect carbapenemase production revealed an overall sensitivity of 98.29% and specificity of 17.95% for the detection of carbapenemase production. For the classification of carbapenemases classes A, B, and D, the PCD correctly classified 79.17% Enterobacterales and 67.16% non-fermentative gram-negative rods. The PCD assay is a reliable tool for the detection of carbapenem resistance and allows for parallel analysis of carbapenemase production. However, while sensitivity is high, low specificity in carbapenemase detection and erroneous classification demands mandatory confirmation by alternative methods, especially in non-fermentative gram-negative bacteria.

Background: The global market for SARS-CoV-2-immunoassays is becoming ever more crowded with antibodytests of various formats, targets and technologies, careful evaluation is crucial for understanding the implications of individual test results. Here, we evaluate the clinical performance of five automated immunoassays on a set of clinical samples. Methods: Serum/plasma samples of 75 confirmed COVID-19 patients and 320 pre-pandemic serum samples of healthy blood donors were subjected to two IgG and three total antibody SARS-CoV-2-immunoassays. All test setups were automated workflows. Results: Positivity of assays (onset of symptoms > 10 days) ranged between 68.4 % and 81.6 % (Diasorin 68.4 %, Euroimmun 70.3 %, Siemens 73.7 %, Roche 79.0 % and Wantai 81.6 %). All examined assays demonstrated high specificity of > 99 % (Euroimmun, Diasorin: 99.1 %, Wantai: 99.4 %) but only two reached levels above 99.5 % (Roche: 99.7 %, Siemens 100 %). Interestingly, there was no overlap in false positive results between the assays. The strongest correlation of quantitative results was observed between the Diasorin and Euroimmun IgG tests (r(2) = 0.76). Overall, we observed no difference in the distribution of test results between female and male patients (p-values: 0.18 - 0.87). A significant difference between severely versus critically ill patients was demonstrated for the Euroimmun, Diasorin, Wantai and Siemens assays (p-values:0.041). Conclusion: All assays showed good clinical performance. Our data confirm that orthogonal test strategies as recommended by the CDC can enhance clinical specificity. However, the suboptimal rates of test positivity found at time of hospitalization in this cohort underline the importance of molecular diagnostics to rule out/confirm active infection with SARS-CoV-2.

Analyses of infection chains have demonstrated that severe acute respiratory syndrome coronavirus 2 is highly transmissive. However, data on postmortem stability and infectivity are lacking. Our finding of nasopharyngeal viral RNA stability in 79 corpses showed no time-dependent decrease. Maintained infectivity is supported by virus isolation up to 35 hours postmortem.

OBJECTIVES:To determine the influence of local spread of clonal strains and testing of follow-up isolates on categorical (CA) and essential agreement rates (EA) of common colistin (COL) drug susceptibility testing methods with the broth microdilution (BMD) reference method. METHODS:COL MICs were determined for 178 bacterial isolates (Enterobacteriaceae, n = 97; Pseudomonas aeruginosa, n = 81) collected within one year from 64 patients by BMD according to ISO standard 20776-1 (reference method), the SensiTest BMD panel (ST), agar dilution (AD), the VITEK 2 instrument, and gradient diffusion (GD) using antibiotic strips of two and Muller-Hinton agar plates of three manufacturers. CA and EA with BMD were calculated for all isolates and compared to the subset of 68 unique isolates. RESULTS:CA ranges were 79.4% to 94.1% for the unique isolateq panel and 89.9% to 96.1% for all tested isolates. EA ranges were 64.7% to 86.8% and 67.4% to 91.0%, respectively. In both panels, EA for all GD assays was lower than 90%. Both lower and higher EA values ranging from-18.3% (MTS on BD agar) to + 6.3% (AD, Vitek 2) were observed in the full one-year sample. Acquisition of colistin resistance under therapy was observed for 3 patients. CONCLUSIONS:i) Repeat testing and local spread of clonal strains can positively or negatively affect CA and EA, ii) CA is more robust towards local influences than EA, iii) EA of GD and AD methods for COL with the reference BMD method is insufficient.