Evaluation of a host-protein signature score for differentiating between bacterial and viral infections: real-life evidence from a German tertiary hospital.

PURPOSE: A host-protein signature score, consisting of serum-concentrations of C-reactive protein, tumour necrosis factor-related apoptosis-inducing ligand, and interferon gamma-induced protein 10, was validated for distinguishing between bacterial and viral infections as an antimicrobial stewardship measure for routine clinical practice among adult patients in a German tertiary hospital. METHODS: This single-centre, explorative study prospectively assessed the host-protein signature score, comparing it with serum procalcitonin (PCT) in patients with blood stream infections (BSI) and evaluating its efficacy in patients with viral infections against the standard of care (SOC) to assess the need for antibiotics due to suspected bacterial super/coinfection. Manufacturer-specified threshold scores were used to differentiate viral (< 35) and bacterial (> 65) infections. RESULTS: Ninety-seven patients (BSI [n = 56]; viral infections [n = 41]) were included. The score (cut-off score > 65) tended to detect BSI with higher sensitivity than did PCT (cut-off > 0.5 ng/mL) (87.5% vs. 76.6%). Three patients (5.4%) with BSI had a score < 35. One patient with BSI did not receive antibiotic treatment following SOC prior to positive blood culture results. Among patients with viral infections, 29 (70.7%) had scores > 65, indicating bacterial superinfections. Additionally, 11 patients (26.8%) had scores < 35, indicating no bacterial superinfections. In total, the antibiotic treatment discrepancy in the viral group between the SOC and a host-protein signature score guided approach was 2/41 patients (4.9%). CONCLUSION: The score tended towards a higher sensitivity in detecting BSI than that with PCT. However, its impact on reducing antibiotic use in viral infections was minor compared with that of SOC.

Implementation and clinical evaluation of an Mpox virus laboratory-developed test on a fully automated random-access platform.

While Mpox virus (MPXV) diagnostics were performed in specialized laboratories only, the global emergence of Mpox cases in 2022 revealed the need for a more readily available diagnostic. Automated random-access platforms with fast nucleic acid extraction and PCR have become established in many laboratories, providing faster and more accessible testing. In this study, we adapted a previously published generic MPXV-PCR as a lab-developed test (LDT) on a NeuMoDx Molecular System and isolated MPXV clones from patient materials. To reduce the handling of infectious material, we evaluated a viral lysis buffer (VLB) for sample pretreatment. We further compared the MPXV-LDT-PCR to conventional real-time PCR, determined its sensitivity and specificity using positive swabs, and assessed its performance using external quality assessment samples. Pretreatment of samples with 50% VLB reduced MPXV infectivity by approximately 200-fold while maintaining PCR sensitivity. The assay demonstrated a sensitivity and specificity of 100% with no cross-reactivity in the samples tested and performed with a limit of detection of 262 GE/mL. In summary, the assay had a turnaround time of fewer than 2 h and can easily be transferred to other automated PCR platforms, providing a basis for developing rapid assays for upcoming pandemics.

Tixagevimab/Cilgavimab for COVID-19 Pre-Exposure Prophylaxis in Hematologic Patients-A Tailored Approach Based on SARS-CoV-2 Vaccine Response.

Patients with hematologic malignancies still face a significant risk of severe coronavirus disease 2019 (COVID-19). The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-neutralizing monoclonal antibody combination tixagevimab/cilgavimab (TIX/CGB) could be administered to immunocompromised patients for pre-exposure prophylaxis (PrEP) before the emergence of TIX/CGB-resistant COVID-19 Omicron variants. TIX/CGB application could be carried out regardless of the host's immune response to previous active SARS-CoV-2 vaccinations or infections. Because the efficacy of COVID-19 PrEP remains unclear, especially in SARS-CoV-2-seropositive patients, German national guidelines recommended TIX/CGB PrEP only for SARS-CoV-2-seronegative patients in addition to an intensified active vaccination schedule. Having followed these guidelines, we now report the characteristics and outcomes of 54 recipients of TIX/CGB PrEP in SARS-CoV-2-seronegative patients with hematological disease from a German tertiary medical center and compare them to 125 seropositive patients who did not receive any PrEP. While the number of patients with B-cell lymphomas was significantly higher in the seronegative cohort (33 (61%) vs. 18 (14%) cases, p < 0.01), patients with myeloid diseases were significantly more frequent in the seropositive cohort (51 (41%) vs. 5 (9%) cases, p < 0.01). Strikingly, patients who had undergone allogeneic hematopoietic stem cell transplantation were significantly more likely (forty-nine (39%) vs. six (11%) cases, p < 0.01) to be SARS-CoV-2 seropositive. We observed that prophylactic application of TIX/CGB PrEP to a highly vulnerable group of SARS-CoV-2-seronegative patients resulted in a similar number of COVID-19 breakthrough infections compared to the untreated seropositive control group (16 (32%) vs. 39 (36%), p = 0.62) and comparable COVID-19-related outcomes like hospitalization and oxygen requirement throughout an extended follow-up period of 12 months. In conclusion, our results support the tailored approach of administering TIX/CGB PrEP only to SARS-CoV-2-seronegative patients during the COVID-19 pandemic and might provide a rationale for similar strategies during future outbreaks/diseases, especially in times of initial limited availability and/or financial constraints.

Postmortem Minimally Invasive Autopsy in Critically Ill COVID-19 Patients at the Bedside: A Proof-of-Concept Study at the ICU.

BACKGROUND: Economic restrictions and workforce cuts have continually challenged conventional autopsies. Recently, the COVID-19 pandemic has added tissue quality and safety requirements to the investigation of this disease, thereby launching efforts to upgrade autopsy strategies. METHODS: In this proof-of-concept study, we performed bedside ultrasound-guided minimally invasive autopsy (US-MIA) in the ICU of critically ill COVID-19 patients using a structured protocol to obtain non-autolyzed tissue. Biopsies were assessed for their quality (vitality) and length of biopsy (mm) and for diagnosis. The efficiency of the procedure was monitored in five cases by recording the time of each step and safety issues by swabbing personal protective equipment and devices for viral contamination. FINDINGS: Ultrasound examination and tissue procurement required a mean time period of 13 min and 54 min, respectively. A total of 318 multiorgan biopsies were obtained from five patients. Quality and vitality standards were fulfilled, which not only allowed for specific histopathological diagnosis but also the reliable detection of SARS-CoV-2 virions in unexpected organs using electronic microscopy and RNA-expressing techniques. INTERPRETATION: Bedside multidisciplinary US-MIA allows for the fast and efficient acquisition of autolytic-free tissue and offers unappreciated potential to overcome the limitations of research in postmortem studies.

Clinical and immunological benefits of full primary COVID-19 vaccination in individuals with SARS-CoV-2 breakthrough infections: A prospective cohort study in non-hospitalized adults.

BACKGROUND: SARS-CoV-2 variants of concern (VOC) may result in breakthrough infections (BTIs) in vaccinated individuals. The aim of this study was to investigate the effects of full primary (two-dose) COVID-19 vaccination with wild-type-based SARS-CoV-2 vaccines on symptoms and immunogenicity of SARS-CoV-2 VOC BTIs. METHODS: In a longitudinal multicenter controlled cohort study in Bavaria, Germany, COVID-19 vaccinated and unvaccinated non-hospitalized individuals were prospectively enrolled within 14 days of a PCR-confirmed SARS-CoV-2 infection. Individuals were visited weekly up to 4 times, performing a structured record of medical data and viral load assessment. SARS-CoV-2-specific antibody response was characterized by anti-spike-(S)- and anti-nucleocapsid-(N)-antibody concentrations, anti-S-IgG avidity and neutralization capacity. RESULTS: A total of 300 individuals (212 BTIs, 88 non-BTIs) were included with VOC Alpha or Delta SARS-CoV-2 infections. Full primary COVID-19 vaccination provided a significant effectiveness against five symptoms (relative risk reduction): fever (33 %), cough (21 %), dysgeusia (22 %), dizziness (52 %) and nausea/vomiting (48 %). Full primary vaccinated individuals showed significantly higher 50 % inhibitory concentration (IC50) values against the infecting VOC compared to unvaccinated individuals at week 1 (269 vs. 56, respectively), and weeks 5-7 (1,917 vs. 932, respectively) with significantly higher relative anti-S-IgG avidity (78% vs. 27 % at week 4, respectively). CONCLUSIONS: Full primary COVID-19 vaccination reduced symptom frequencies in non-hospitalized individuals with BTIs and elicited a more rapid and longer lasting neutralization capacity against the infecting VOC compared to unvaccinated individuals. These results support the recommendation to offer at least full primary vaccination to all adults to reduce disease severity caused by immune escape-variants.

Improved detection of infection with SARS-CoV-2 Omicron variants of concern in healthcare workers by a second-generation rapid antigen test.

IMPORTANCE: The results from this study demonstrate the usefulness of a second-generation rapid antigen test for early detection of infection with the SARS-CoV-2 Omicron variant of concern (VoC) and reveal a higher sensitivity to detect immune escape Omicron VoCs compared to a first-generation rapid antigen test (89.4% vs 83.7%) in the high-risk group of healthcare workers.

RBD-Based ELISA and Luminex Predict Anti-SARS-CoV-2 Surrogate-Neutralizing Activity in Two Longitudinal Cohorts of German and Spanish Health Care Workers.

The ability of antibodies to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important correlate of protection. For routine evaluation of protection, however, a simple and cost-efficient anti-SARS-CoV-2 serological assay predictive of serum neutralizing activity is needed. We analyzed clinical epidemiological data and blood samples from two cohorts of health care workers in Barcelona and Munich to compare several immunological readouts for evaluating antibody levels that could be surrogates of neutralizing activity. We measured IgG levels against SARS-CoV-2 spike protein (S), its S2 subunit, the S1 receptor binding domain (RBD), and the full length and C terminus of nucleocapsid (N) protein by Luminex, and against RBD by enzyme-linked immunosorbent assay (ELISA), and assessed those as predictors of plasma surrogate-neutralizing activity measured by a flow cytometry assay. In addition, we determined the clinical and demographic factors affecting plasma surrogate-neutralizing capacity. Both cohorts showed a high positive correlation between IgG levels to S antigen, especially to RBD, and the levels of plasma surrogate-neutralizing activity, suggesting RBD IgG as a good correlate of plasma neutralizing activity. Symptomatic infection, with symptoms such as loss of taste, dyspnea, rigors, fever and fatigue, was positively associated with anti-RBD IgG positivity by ELISA and Luminex, and with plasma surrogate-neutralizing activity. Our serological assays allow for the prediction of serum neutralization activity without the cost, hazards, time, and expertise needed for surrogate or conventional neutralization assays. Once a cutoff is established, these relatively simple high-throughput antibody assays will provide a fast and cost-effective method of assessing levels of protection from SARS-CoV-2 infection. IMPORTANCE Neutralizing antibody titers are the best correlate of protection against SARS-CoV-2. However, current tests to measure plasma or serum neutralizing activity do not allow high-throughput screening at the population level. Serological tests could be an alternative if they are proved to be good predictors of plasma neutralizing activity. In this study, we analyzed the SARS-CoV-2 serological profiles of two cohorts of health care workers by applying Luminex and ELISA in-house serological assays. Correlations of both serological tests were assessed between them and with a flow cytometry assay to determine plasma surrogate-neutralizing activity. Both assays showed a high positive correlation between IgG levels to S antigens, especially RBD, and the levels of plasma surrogate-neutralizing activity. This result suggests IgG to RBD as a good correlate of plasma surrogate-neutralizing activity and indicates that serology of IgG to RBD could be used to assess levels of protection from SARS-CoV-2 infection.

Dynamics of spike-and nucleocapsid specific immunity during long-term follow-up and vaccination of SARS-CoV-2 convalescents.

Anti-viral immunity continuously declines over time after SARS-CoV-2 infection. Here, we characterize the dynamics of anti-viral immunity during long-term follow-up and after BNT162b2 mRNA-vaccination in convalescents after asymptomatic or mild SARS-CoV-2 infection. Virus-specific and virus-neutralizing antibody titers rapidly declined in convalescents over 9 months after infection, whereas virus-specific cytokine-producing polyfunctional T cells persisted, among which IL-2-producing T cells correlated with virus-neutralizing antibody titers. Among convalescents, 5% of individuals failed to mount long-lasting immunity after infection and showed a delayed response to vaccination compared to 1% of naïve vaccinees, but successfully responded to prime/boost vaccination. During the follow-up period, 8% of convalescents showed a selective increase in virus-neutralizing antibody titers without accompanying increased frequencies of circulating SARS-CoV-2-specific T cells. The same convalescents, however, responded to vaccination with simultaneous increase in antibody and T cell immunity revealing the strength of mRNA-vaccination to increase virus-specific immunity in convalescents.

Dynamics of humoral and cellular immune responses after homologous and heterologous SARS-CoV-2 vaccination with ChAdOx1 nCoV-19 and BNT162b2.

BACKGROUND: Vaccines are an important means to overcome the SARS-CoV-2 pandemic. They induce specific antibody and T-cell responses but it remains open how well vaccine-induced immunity is preserved over time following homologous and heterologous immunization regimens. Here, we compared the dynamics of humoral and cellular immune responses up to 180 days after homologous or heterologous vaccination with either ChAdOx1-nCoV-19 (ChAd) or BNT162b2 (BNT) or both. METHODS: Various tests were used to determine the humoral and cellular immune response. To quantify the antibody levels, we used the surrogate neutralization (sVNT) assay from YHLO, which we augmented with pseudo- and real virus neutralization tests (pVNT and rVNT). Antibody avidity was measured by a modified ELISA. To determine cellular reactivity, we used an IFN-γ Elispot, IFN-γ/IL Flurospot, and intracellular cytokine staining. FINDINGS: Antibody responses significantly waned after vaccination, irrespective of the regimen. The capacity to neutralize SARS-CoV-2 - including variants of concern such as Delta or Omicron - was superior after heterologous compared to homologous BNT vaccination, both of which resulted in longer-lasting humoral immunity than homologous ChAd immunization. All vaccination regimens induced stable, polyfunctional T-cell responses. INTERPRETATION: These findings demonstrate that heterologous vaccination with ChAd and BNT is a potent alternative to induce humoral and cellular immune protection in comparison to the homologous vaccination regimens. FUNDING: The study was funded by the German Centre for Infection Research (DZIF), the European Union's "Horizon 2020 Research and Innovation Programme" under grant agreement No. 101037867 (VACCELERATE), the "Bayerisches Staatsministerium für Wissenschaft und Kunst" for the CoVaKo-2021 and the For-COVID projects and the Helmholtz Association via the collaborative research program "CoViPa". Further support was obtained from the Federal Ministry of Education and Science (BMBF) through the "Netzwerk Universitätsmedizin", project "B-Fast" and "Cov-Immune". KS is supported by the German Federal Ministry of Education and Research (BMBF, 01KI2013) and the Else Kröner-Stiftung (2020_EKEA.127).

Three exposures to the spike protein of SARS-CoV-2 by either infection or vaccination elicit superior neutralizing immunity to all variants of concern.

Infection-neutralizing antibody responses after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or coronavirus disease 2019 vaccination are an essential component of antiviral immunity. Antibody-mediated protection is challenged by the emergence of SARS-CoV-2 variants of concern (VoCs) with immune escape properties, such as omicron (B.1.1.529), which is rapidly spreading worldwide. Here we report neutralizing antibody dynamics in a longitudinal cohort of coronavirus disease 2019 convalescent and infection-naive individuals vaccinated with mRNA BNT162b2 by quantifying SARS-CoV-2 spike protein antibodies and determining their avidity and neutralization capacity in serum. Using live-virus neutralization assays, we show that a superior infection-neutralizing capacity against all VoCs, including omicron, developed after either two vaccinations in convalescents or a third vaccination or breakthrough infection of twice-vaccinated, naive individuals. These three consecutive spike antigen exposures resulted in an increasing neutralization capacity per anti-spike antibody unit and were paralleled by stepwise increases in antibody avidity. We conclude that an infection-plus-vaccination-induced hybrid immunity or a triple immunization can induce high-quality antibodies with superior neutralization capacity against VoCs, including omicron.