Surrogate Virus Neutralisation Test Based on Nanoluciferase-Tagged Antigens to Quantify Inhibitory Antibodies against SARS-CoV-2 and Characterise Omicron-Specific Reactivity in a Vaccination Cohort.

Virus-specific antibodies are crucial for protective immunity against SARS-CoV-2. Assessing functional antibodies through conventional or pseudotyped virus neutralisation tests (pVNT) requires high biosafety levels. Alternatively, the virus-free surrogate virus neutralisation test (sVNT) quantifies antibodies interfering with spike binding to angiotensin-converting enzyme 2. We evaluated secreted nanoluciferase-tagged spike protein fragments as diagnostic antigens in the sVNT in a vaccination cohort. Initially, spike fragments were tested in a capture enzyme immunoassay (EIA), identifying the receptor binding domain (RBD) as the optimal diagnostic antigen. The sensitivity of the in-house sVNT applying the nanoluciferase-labelled RBD equalled or surpassed that of a commercial sVNT (cPass, GenScript Diagnostics) and an in-house pVNT four weeks after the first vaccination (98% vs. 94% and 72%, respectively), reaching 100% in all assays four weeks after the second and third vaccinations. When testing serum reactivity with Omicron BA.1 spike, the sVNT and pVNT displayed superior discrimination between wild-type- and variant-specific serum reactivity compared to a capture EIA. This was most pronounced after the first and second vaccinations, with the third vaccination resulting in robust, cross-reactive BA.1 construct detection. In conclusion, utilising nanoluciferase-labelled antigens permits the quantification of SARS-CoV-2-specific inhibitory antibodies. Designed as flexible modular systems, the assays can be readily adjusted for monitoring vaccine efficacy.

Impaired humoral immunity to BQ.1.1 in convalescent and vaccinated patients.

Determining SARS-CoV-2 immunity is critical to assess COVID-19 risk and the need for prevention and mitigation strategies. We measured SARS-CoV-2 Spike/Nucleocapsid seroprevalence and serum neutralizing activity against Wu01, BA.4/5 and BQ.1.1 in a convenience sample of 1,411 patients receiving medical treatment in the emergency departments of five university hospitals in North Rhine-Westphalia, Germany, in August/September 2022. 62% reported underlying medical conditions and 67.7% were vaccinated according to German COVID-19 vaccination recommendations (13.9% fully vaccinated, 54.3% one booster, 23.4% two boosters). We detected Spike-IgG in 95.6%, Nucleocapsid-IgG in 24.0%, and neutralization against Wu01, BA.4/5 and BQ.1.1 in 94.4%, 85.0%, and 73.8% of participants, respectively. Neutralization against BA.4/5 and BQ.1.1 was 5.6- and 23.4-fold lower compared to Wu01. Accuracy of S-IgG detection for determination of neutralizing activity against BQ.1.1 was reduced substantially. We explored previous vaccinations and infections as correlates of BQ.1.1 neutralization using multivariable and Bayesian network analyses. Given a rather moderate adherence to COVID-19 vaccination recommendations, this analysis highlights the need to improve vaccine-uptake to reduce the COVID-19 risk of immune evasive variants. The study was registered as clinical trial (DRKS00029414).

Parvovirus B19 infection in pediatric allogeneic hematopoietic cell transplantation - Single-center experience and review.

BACKGROUND: Parvovirus B19 (B19V) infection following pediatric hematopoietic cell transplantation (HCT) is a rare complication and available data is scarce. Therefore, we present the experience with B19V Infection in allogeneic pediatric HCT recipients at our transplant center together with a systematic review of the literature. METHODS: Pediatric HCT patients with Parvovirus B19 infection treated at the University Children's Hospital Münster between 1999 and 2021 were retrospectively identified and clinical data were analyzed. Additionally, a systematic MEDLINE search to identify relevant articles was performed. RESULTS: We identified three out of 445 patients (0.6%) with B19V infection post-transplantation. B19V infection occurred in combination with other complications like Graft-versus-Host disease, additional infections, or autoimmune-mediated hemolysis potentially triggered by B19V. In one patient these complications lead to a fatal outcome. The review of the literature showed considerable morbidity of B19V infection with the potential for life-threatening complications. Most patients were treated by red blood cell transfusion and intravenous immunoglobulins (IVIG) with a high succession rate. CONCLUSION: Symptomatic B19V infection following HCT remains a rare but potentially challenging complication. A causal antiviral therapy does not exist as well as general recommendations on dosage and duration of IVIG therapy. Despite this, most patients are treated successfully with these measures. Additionally, transmission via blood or stem cell products is also rare and no general recommendations on B19V screenings exist.

Letermovir for Prophylaxis and Pre-emptive Therapy of Cytomegalovirus Infection in Paediatric Allogeneic Haematopoietic Cell Transplant Patients.

BACKGROUND: Cytomegalovirus (CMV) infection is a frequent event in patients undergoing allogeneic haematopoietic cell transplantation (HCT) and is associated with increased morbidity and mortality due to eventual progress to end-organ disease. Letermovir prophylaxis for CMV infections has become a standard of care in adult HCT recipients due to its efficacy and high tolerability. However, it is not yet approved for paediatric patients. OBJECTIVE: In a retrospective single-centre observational study we evaluated the use of letermovir for prophylaxis or pre-emptive treatment of cytomegalovirus (CMV) infection in seropositive paediatric HCT recipients receiving the compound outside of clinical trials. The primary endpoint was CMV reactivation requiring a change of medication. METHODS: A total of 17 patients (seven female/ten male; median age 12.2 [range 3.5-19] years, median body weight 39.5 [range 15-63] kg; median follow-up time 463.7 [range 41-1022] days) were identified who were started on oral (14) or intravenous (3) followed by oral (2) letermovir shortly after neutrophil engraftment at doses determined on the basis of age, weight, and concomitant cyclosporine use. RESULTS: Five patients had no evidence of viral replication (prophylactic use), while 12 patients had varying extents of viral replication (pre-emptive therapy). A change of therapy was required in one patient due to a sustained increase in CMV viral load, and in two patients, letermovir was stopped without later reactivation after initiation of palliative care for recurrent leukaemia. Of the 14 patients who completed treatment, 3 had evidence of transient viral replication after end of treatment that required no further antiviral treatment. No patients (of 17) discontinued letermovir due to an adverse event. CONCLUSION: Letermovir was effective in controlling CMV infection in seropositive paediatric allogeneic HCT recipients and was overall well tolerated. Pending completion of the still ongoing paediatric investigation plans, letermovir will be an important adjunct to our options for control of infectious complications in this special population.

COVID-19 rapid molecular point-of-care testing is effective and cost-beneficial for the acute care of trauma patients.

PURPOSE: To evaluate the accuracy and cost benefit of a rapid molecular point-of-care testing (POCT) device detecting COVID-19 within a traumatological emergency department. BACKGROUND: Despite continuous withdrawal of COVID-19 restrictions, hospitals will remain particularly vulnerable to local outbreaks which is reflected by a higher institution-specific basic reproduction rate. Patients admitted to the emergency department with unknown COVID-19 infection status due to a- or oligosymptomatic COVID-19 infection put other patients and health care workers at risk, while fast diagnosis and treatment is necessary. Delayed testing results in additional costs to the health care system. METHODS: From the 8th of April 2021 until 31st of December 2021, all patients admitted to the emergency department were tested with routine RT-PCR and rapid molecular POCT device (Abbott ID NOW™ COVID-19). COVID-19-related additional costs for patients admitted via shock room or emergency department were calculated based on internal cost allocations. RESULTS: 1133 rapid molecular tests resulted in a sensitivity of 83.3% (95% CI 35.9-99.6%), specificity of 99.8% (95% CI 99.4-100%), a positive predictive value of 71.4% (95% CI 29-96.3%) and a negative predictive value of 99.9% (95% CI 99.5-100%) as compared to RT-PCR. Without rapid COVID-19 testing, each emergency department and shock room admission with subsequent surgery showed additional direct costs of 2631.25€, without surgery of 729.01€. CONCLUSION: Although rapid molecular COVID-19 testing can initially be more expensive than RT-PCR, subsequent cost savings, improved workflows and workforce protection outweigh this effect by far. The data of this study support the use of a rapid molecular POCT device in a traumatological emergency department.

Efficacy of COVID-19 Booster Vaccines in Patients with Hematologic Malignancies: Experiences in a Real-World Scenario.

Background: Two-dose COVID-19 vaccination often results in poor humoral response rates in patients with hematologic malignancies (HMs); yet responses to COVID-19 booster vaccines and the risk of COVID-19 infection post-booster are mostly uncertain. Methods: We included 200 outpatients with HMs and predominantly lymphoid neoplasms (96%, 191/200) in our academic center and reported on the humoral responses, which were assessed by measurement of anti-spike IgG antibodies in peripheral blood as early as 14 days after mRNA-based prime-boost vaccination, as well as factors hampering booster efficacy. Previous basic (double) immunization was applied according to the local recommendations with mRNA- and/or vector-based vaccines. We also report on post-booster COVID-19 breakthrough infections that emerged in the Omicron era and the prophylaxis strategies that were applied to poor and non-responders to booster vaccines. Results: A total of 55% (110/200) of the patients achieved seroconversion (i.e., anti-spike protein IgG antibody titer > 100 AU/mL assessed in median 48 days after prime-boost vaccination) after prime-boost vaccination. Multivariable analyses revealed age, lymphocytopenia, ongoing treatment and prior anti-CD20 B-cell depletion to be independent predictors for booster failure. With each month between anti-CD20-mediated B-cell depletion and booster vaccination, the probability of seroconversion increased by approximately 4% (p < 0.001) and serum−antibody titer (S-AbT) levels increased by 90 AU/mL (p = 0.011). Notably, obinutuzumab treatment was associated with an 85% lower probability for seroconversion after prime-boost vaccination compared to rituximab (p = 0.002). Of poor or non-responders to prime-boost vaccination, 41% (47/114) underwent a second booster and 73% (83/114) underwent passive immunization. COVID-19 breakthrough infections were observed in 15% (29/200) of patients after prime-boost vaccination with predominantly mild courses (93%). Next to seroconversion, passive immunization was associated with a significantly lower risk of COVID-19 breakthrough infections after booster, even in vaccine non-responders (all p < 0.05). In a small proportion of analyzed patients with myeloid neoplasms (9/200), the seroconversion rate was higher compared to those with lymphoid ones (78% vs. 54%, accordingly), while the incidence rate of COVID-19 breakthrough infections was similar (22% vs. 14%, respectively). Following the low frequency of myeloid neoplasms in this study, the results may not be automatically applied to a larger cohort. Conclusions: Patients with HMs are at a high risk of COVID-19 booster vaccine failure; yet COVID-19 breakthrough infections after prime-boost vaccination are predominantly mild. Booster failure can likely be overcome by passive immunization, thereby providing immune protection against COVID-19 and attenuating the severity of COVID-19 courses. Further sophistication of clinical algorithms for preventing post-vaccination COVID-19 breakthrough infections is urgently needed.

Broadly Applicable, Virus-Free Dual Reporter Assay to Identify Compounds Interfering with Membrane Fusion: Performance for HSV-1 and SARS-CoV-2.

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell-cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell-cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell-cell fusion in Vero cells.

Assessment of Neonatal Cord Blood SARS-CoV-2 Antibodies after COVID-19 Vaccination in Pregnancy: A Prospective Cohort Study.

Introduction Maternally derived antibodies are a key element of neonatal immunity. So far, limited data has shown transplacental transmission of antibodies after coronavirus disease 2019 (COVID-19) vaccination with BNT162b2 in the third trimester. Our aim was to detect vertically transferred immunity after COVID-19 vaccination with BNT162b2 (Comirnaty, BioNTech-Pfizer) or mRNA-1273 (Spikevax, Moderna) in the first, second or third trimester of pregnancy, and investigate the impact of maternal characteristics on umbilical cord antibody titre in newborns after delivery. Study Design Women who gave birth in our department and were vaccinated against COVID-19 during pregnancy were enrolled in CRONOS Satellite, a subproject of the German COVID-19-Related Obstetric and Neonatal Outcome Study. The titre of immunoglobulin G (IgG) antibodies to the receptor-binding domain of the SARS-CoV-2 spike protein was quantified in umbilical cord blood using the SARS-CoV-2 IgG II Quant immunoassay. Correlations between antibody titre and variables, including week of pregnancy when vaccinated, interval between vaccination and delivery, age and body mass index (BMI) were assessed with Spearman's rank correlation. A follow-up was conducted by phone interview 4 - 6 weeks after delivery. Results The study cohort consisted of 70 women and their 74 newborns. Vaccine-generated antibodies were present in all samples, irrespective of the vaccination type or time of vaccination. None of the parameters of interest showed a meaningful correlation with cord blood antibody concentrations (rho values < 0.5). No adverse outcomes (including foetal malformation) were reported, even after vaccination in the first trimester. Conclusions Transplacental passage of SARS-CoV-2 antibodies from mother to child was demonstrated in all cases in the present study. It can therefore be assumed that the newborns of mothers vaccinated at any time during pregnancy receive antibodies via the placenta which potentially provide them with protection against COVID-19. This is an additional argument when counselling pregnant women about vaccination in pregnancy.

Humoral Immune Response in IBD Patients Three and Six Months after Vaccination with the SARS-CoV-2 mRNA Vaccines mRNA-1273 and BNT162b2.

Severe acute respiratory syndrome coronovirus-2 (SARS-CoV-2) is the cause of the coronavirus disease 2019 (COVID-19) pandemic. Vaccination is considered the core approach to containing the pandemic. There is currently insufficient evidence on the efficacy of these vaccines in immunosuppressed inflammatory bowel disease (IBD) patients. The aim of this study was to investigate the humoral response in immunosuppressed IBD patients after COVID-19 mRNA vaccination. In this prospective study, IgG antibody levels (AB) against the SARS-CoV-2 receptor-binding domain (spike-protein) were quantitatively determined. For assessing the potential neutralizing capacity, a SARS-CoV-2 surrogate neutralization test (sVNT) was employed in IBD patients (n = 95) and healthy controls (n = 38). Sera were examined prior to the first/second vaccination and 3/6 months after second vaccination. Patients showed lower sVNT (%) and IgG-S (AU/mL) AB both before the second vaccination (sVNT p < 0.001; AB p < 0.001) and 3 (sVNT p = 0.002; AB p = 0.001) and 6 months (sVNT p = 0.062; AB p = 0.061) after the second vaccination. Although seroconversion rates (sVNT, IgG-S) did not differ between the two groups 3 months after second vaccination, a significant difference was seen 6 months after second vaccination (sVNT p = 0.045). Before and three months after the second vaccination, patients treated with anti-tumor necrosis factor (TNF) agents showed significantly lower AB than healthy subjects. In conclusion, an early booster shot vaccination should be discussed for IBD patients on anti-TNF therapy.

Phenotypic Variants of Bacterial Colonies in Microbiological Diagnostics: How Often Are They Indicative of Differing Antimicrobial Susceptibility Patterns?

Phenotypic variants (PV) are colonies of the same species in the same specimen with different morphological features. It is controversial whether antimicrobial susceptibility testing (AST) should be done for all PV. The objectives of this study were to quantify the proportion of differing antimicrobial susceptibility patterns (dASP) among PV and to identify species and antimicrobial compounds that are mostly affected. All PV from routine diagnostics (University Hospital Münster, Germany; 1 September 2019 to 31 August 2020) were subjected to species identification (matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS]) and AST (Vitek 2). To assess the dASP, only antimicrobial agents were considered for which Vitek-derived MIC were available (interpreted according to the EUCAST clinical breakpoints). The categorical agreement (CA; agreement with the AST categories S [susceptible, standard dosing regimen], I [susceptible, increased exposure], R [resistant]) of the PV was calculated. The PV of Escherichia coli (n = 260), Pseudomonas aeruginosa (n = 86), Klebsiella pneumoniae (n = 47), Enterobacter cloacae complex (n = 45), and Staphylococcus aureus (n = 38) were included. The median CA was 95% (range, 80 to 100%, depending on the species). The colony characteristics (e.g., form/size, color, margin, hemolysis) were not indicative for dASP. PV showed a high categorical agreement in the AST categories. This observation supports a test strategy to perform AST for only one colony of PV. IMPORTANCE Phenotypic variants of bacteria are frequent in routine diagnostics and can display differing antimicrobial susceptibility patterns. We found that the likelihood of different antimicrobial susceptibility is low among PV. To save laboratory resources, only one isolate per PV could be tested to guide the antimicrobial treatment of patients.

Healthcare-Associated SARS-CoV-2 Transmission-Experiences from a German University Hospital.

During the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, healthcare systems worldwide have to prevent nosocomial SARS-CoV-2 transmission while maintaining duty of care. In our study, we characterize the transmission dynamic of SARS-CoV-2 in inpatients and healthcare workers (HCWs) at the University Hospital Münster (UHM) in northwest Germany. We identified 27 cases of healthcare-associated SARS-CoV-2 infections (4 inpatients and 23 HCWs) who had contact with patients and/or HCWs without the use of adequate PPE. The contacts of these index cases were followed up for SARS-CoV-2 infection after unprotected exposure and a quantitative measure of probability of becoming infected, the attack rate, was calculated. In addition, transmission was evaluated in the context of infection control measures established during the pandemic and we compared the epidemiological data of all index cases, including symptoms and Ct values of virology test results. The overall attack rate in the hospital setting was 1.3% (inpatients 0.9%, HCWs 1.6%). However, during an outbreak, the attack rate was 25.5% (inpatients 20.0%, HCWs 29.6%). For both scenarios, HCWs had a higher attack rate illustrating their role in healthcare-associated SARS-CoV-2 transmission. Taken together, our experiences demonstrate how infection control measures can minimize the transmission of SARS-CoV-2 in the healthcare setting.

Systemic viral infection in children receiving chemotherapy for acute leukemia.

Systemic viral diseases frequently occur in allogeneic hematopoietic stem cell transplantation, but data in children receiving chemotherapy for acute leukemia are scarce. We therefore collected and analyzed the published data on symptomatic infection from cytomegalovirus, herpes simplex virus, varicella zoster virus, parvovirus B19, or adenovirus in pediatric acute leukemia. Reports on 68 children were identified, of whom 16 patients have died from the infection. Further studies have to (1) evaluate the true incidence of these infections in pediatric acute leukemia, (2) their impact on outcome, and (3) whether a subpopulation of patients could benefit from screening and prophylactic strategies.