Antibodies in healthcare personnel following severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) infection.

In a prospective cohort of healthcare personnel (HCP), we measured severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) nucleocapsid IgG antibodies after SARS-CoV-2 infection. Among 79 HCP, 68 (86%) were seropositive 14-28 days after their positive PCR test, and 54 (77%) of 70 were seropositive at the 70-180-day follow-up. Many seropositive HCP (95%) experienced an antibody decline by the second visit.

Implementation of high-sensitivity troponin with a rapid diagnostic algorithm reduces emergency department length of stay for discharged patients.

INTRODUCTION: High sensitivity troponin (hs-cTn) and diagnostic algorithms are used to rapidly triage patients with symptoms of acute myocardial infarction in emergency departments (ED). However, few studies have evaluated the impact of simultaneously implementing hs-cTn and a rapid rule-out algorithm on length of stay (LOS). METHODS: We assessed the impact of transitioning from contemporary cTnI to hs-cTnI in 59,232 ED encounters over three years. hs-cTnI was implemented with an orderable series that included baseline, two-, four-, and six-hour specimens collected at provider discretion and operationalized with an algorithm to calculate the change in hs-cTnI from baseline and provide interpretations of "insignificant", "significant," or "equivocal." Patient demographics, results, chief complaint, disposition, and ED LOS were captured from the electronic medical record. RESULTS: cTnI was ordered for 31,875 encounters prior to hs-cTnI implementation and 27,357 after. The proportion of cTnI results above the 99th percentile upper reference limit decreased from 35.0% to 27.0% for men and increased from 27.8% to 34.8% for women. Among discharged patients, the median LOS decreased by 0.6 h (0.5-0.7). LOS among discharged patients with a chief complaint of chest pain decreased by 1.0 h (0.8-1.1) and further decreased by 1.2 h (1.0-1.3) if the initial hs-cTnI was below the limit of quantitation. The rate of acute coronary syndrome upon re-presentation within 30 days did not change post-implementation (0.10% versus 0.07%). CONCLUSION: Implementation of an hs-cTnI assay with a rapid rule-out algorithm decreased ED LOS among discharged patients, particularly among those with a chief complaint of chest pain.

Qualitative and quantitative detection of SARS-CoV-2 antibodies from dried blood spots.

INTRODUCTION: Dried blood spot (DBS) sampling is a minimally invasive method for specimen collection with potential multifaceted uses, particularly for serosurveillance of previous SARS-CoV-2 infection. In this study, we assessed DBS as a potential specimen type for assessing IgG and total (including IgG and IgM) antibodies to SARS-CoV-2 in vaccinated and naturally infected patients. METHODS: Six candidate buffers were assessed for eluting blood from DBS cards. The study utilized one hundred and five paired plasma specimens and DBS specimens from prospectively collected SARS-CoV-2 vaccinated individuals, remnants from those with PCR confirmed SARS-CoV-2 infections, or remnants from those without history of infection or vaccination. All specimens were tested with the Siemens SARS-CoV-2 total assay (COV2T) or IgG assay (sCOVG). RESULTS: The lowest backgrounds were observed with water and PBS, and water was used for elution. Relative to plasma samples, DBS samples had a positive percent agreement (PPA) of 94.4% (95% CI: 94.9-100%) for COV2T and 79.2 (68.4-87.0) for sCOVG using the manufacturer's cutoff. The NPA was 100 % (87.1-100.0 and 85.13-100) for both assays. Dilution studies revealed 100% (95% CI: 90.8-100%) qualitative agreement between specimen types on the COV2T assay and 98.0% (88.0-99.9%) with the sCOVG using study defined cutoffs. CONCLUSION: DBS specimens demonstrated high PPA and NPA relative to plasma for SARS-CoV-2 serological testing. Our data support feasibility of DBS sampling for SARS-CoV-2 serological testing.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.

Longitudinal analysis of risk factors associated with severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection among hemodialysis patients and healthcare personnel in outpatient hemodialysis centers

In this prospective, longitudinal study, we examined the risk factors for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection among a cohort of chronic hemodialysis (HD) patients and healthcare personnel (HCPs) over a 6-month period. The risk of SARS-CoV-2 infection among HD patients and HCPs was consistently associated with a household member having SARS-CoV-2 infection.

Antibodies in healthcare personnel following severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) infection

In a prospective cohort of healthcare personnel (HCP), we measured severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) nucleocapsid IgG antibodies after SARS-CoV-2 infection. Among 79 HCP, 68 (86%) were seropositive 14–28 days after their positive PCR test, and 54 (77%) of 70 were seropositive at the 70–180-day follow-up. Many seropositive HCP (95%) experienced an antibody decline by the second visit.

Longitudinal analysis of risk factors associated with severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection among hemodialysis patients and healthcare personnel in outpatient hemodialysis centers.

In this prospective, longitudinal study, we examined the risk factors for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection among a cohort of chronic hemodialysis (HD) patients and healthcare personnel (HCPs) over a 6-month period. The risk of SARS-CoV-2 infection among HD patients and HCPs was consistently associated with a household member having SARS-CoV-2 infection.

Plasmonic Fluor-Enhanced Antigen Arrays for High-Throughput, Serological Studies of SARS-CoV-2.

Serological testing for acute infection or prior exposure is critical for patient management and coordination of public health decisions during outbreaks. Current methods have several limitations, including variable performance, relatively low analytical and clinical sensitivity, and poor detection due to antigenic drift. Serological methods for SARS-CoV-2 detection for the ongoing COVID-19 pandemic suffer from several of these limitations and serves as a reminder of the critical need for new technologies. Here, we describe the use of ultrabright fluorescent reagents, Plasmonic Fluors, coupled with antigen arrays that address a subset of these limitations. We demonstrate its application using patient samples in SARS-CoV-2 serological assays. In our multiplexed assay, SARS-CoV-2 antigens were spotted into 48-plex arrays within a single well of a 96-well plate and used to evaluate remnant laboratory samples of SARS-CoV-2 positive patients. Signal-readout was performed with Auragent Bioscience's Empower microplate reader, and microarray analysis software. Sample volumes of 1 µL were used. High sensitivity of the Plasmonic Fluors combined with the array format enabled us to profile patient serological response to eight distinct SARS-CoV-2 antigens and evaluate responses to IgG, IgM, and IgA. Sensitivities for SARS-CoV-2 antigens during the symptomatic state ranged between 72.5 and 95.0%, specificity between 62.5 and 100%, and the resulting area under the curve values between 0.76 and 0.97. Together, these results highlight the increased sensitivity for low sample volumes and multiplex capability. These characteristics make Plasmonic Fluor-enhanced antigen arrays an attractive technology for serological studies for the COVID-19 pandemic and beyond.

Serial cardiac biomarkers for risk stratification of patients with COVID-19.

OBJECTIVES: Several studies have demonstrated an association between elevated cardiac biomarkers and adverse outcomes in patients with COVID-19. However, the prognostic and predictive capability of a multimarker panel in a prospectively collected, diverse "all-comers" COVID-19 population has not been fully elucidated. DESIGN & METHODS: We prospectively assessed high sensitivity cardiac troponin I (hsTnI), NT-pro B-type Natriuretic Peptide (NT-proBNP), Galectin-3 (Gal-3), and procalcitonin (PCT) in 4,282 serial samples from 358 patients admitted with symptomatic, RT-PCR confirmed SARS-CoV-2 infection. Outcomes examined were 30-day in-hospital mortality and requirement for intubation within 10 days. RESULTS: Baseline hsTnI had the highest AUC for predicting 30-day mortality (0.81; 95% CI, 0.73-0.88), followed by NT-proBNP (0.80; 0.74-0.86), PCT (0.77; 0.70-0.84), and Gal-3 (0.68; 0.60-0.76). HsTnI < 3.5 ng/L at baseline identified patients at low risk for in-hospital mortality (NPV 95.9%, sensitivity 97.3%) and 10-day intubation (NPV 90.4%, sensitivity 88.5%). Continuous, log-2 increases in troponin concentration were associated with reduced survival (p < 0.001) on Kaplan-Meier curves and increased risk of 30-day mortality: HR 1.26 (1.16-1.37) in univariate and 1.19 (1.03-1.4) in multivariate models. Time-varying doubling of concentrations of hsTnI and Gal-3 were associated with increased risk of 30-day mortality (adjusted HR 1.21, 1.06-1.4, and 1.92, 1.40-2.6). CONCLUSION: HsTnI, NT-proBNP, Gal-3, and PCT are elevated at baseline in patients that have worse outcomes from COVID-19. HsTnI was the only independent predictor of 30-day mortality and intubation. Time-varying, doubling in hsTnI and Gal-3 further aided in prognostication of adverse outcomes. These results support the use of hsTnI for triaging patients with COVID-19.

Predictors of humoral response to SARS-CoV-2 mRNA vaccine BNT162b2 in patients receiving maintenance dialysis.

Objective: Patients on dialysis are at high risk for severe COVID-19 and associated morbidity and mortality. We examined the humoral response to SARS-CoV-2 mRNA vaccine BNT162b2 in a maintenance dialysis population. Design: Single-center cohort study. Setting and participants: Adult maintenance dialysis patients at 3 outpatient dialysis units of a large academic center. Methods: Participants were vaccinated with 2 doses of BNT162b2, 3 weeks apart. We assessed anti-SARS-CoV-2 spike antibodies (anti-S) ∼4-7 weeks after the second dose and evaluated risk factors associated with insufficient response. Definitions of antibody response are as follows: nonresponse (anti-S level, <50 AU/mL), low response (anti-S level, 50-839 AU/mL), and sufficient response (anti-S level, ≥840 AU/mL). Results: Among the 173 participants who received 2 vaccine doses, the median age was 60 years (range, 28-88), 53.2% were men, 85% were of Black race, 86% were on in-center hemodialysis and 14% were on peritoneal dialysis. Also, 7 participants (4%) had no response, 27 (15.6%) had a low response, and 139 (80.3%) had a sufficient antibody response. In multivariable analysis, factors significantly associated with insufficient antibody response included end-stage renal disease comorbidity index score ≥5 and absence of prior hepatitis B vaccination response. Conclusions: Although most of our study participants seroconverted after 2 doses of BNT162b2, 20% of our cohort did not achieve sufficient humoral response. Our findings demonstrate the urgent need for a more effective vaccine strategy in this high-risk patient population and highlight the importance of ongoing preventative measures until protective immunity is achieved.

Use of laboratory data for illicit drug use surveillance and identification of socioeconomic risk factors.

BACKGROUND: Drug overdose is the leading cause of death among people 25-44 years of age in the United States. Existing drug surveillance methods are important for prevention and directing treatment, but are limited by delayed reporting and lack of geographic granularity. METHODS: Laboratory urine drug screen and complete metabolic panel data from patients presenting to the emergency department was used to observe long-term and short-term temporal and geospatial changes at the zip code-level in St. Louis. Multivariate linear regression was performed to investigate associations between zip code-level socioeconomic factors and drug screening positivity rates. RESULTS: An increase in the fentanyl positive drug screens was seen during the initial COVID-19 shutdown period in the spring of 2020. A decrease in cocaine positivity was seen in the fall and winter of 2020, with a return to baseline coinciding with the second major COVID-19 shutdown in the summer of 2021. These changes appeared to be independent of changes in emergency department utilization as measured by complete metabolic panels ordered. Significant short-term changes in fentanyl and cocaine positivity rates between specific time periods were able to be localized to individual zip codes. Zip code-level multivariate analysis demonstrated independent associations between socioeconomic/demographic factors and fentanyl/cocaine positivity rates as determined by laboratory drug screening data. CONCLUSIONS: Analyzing clinical laboratory drug screening data can enable a more temporally and geographically granular view of population-level drug use surveillance. Additionally, laboratory data can be utilized to find population-level socioeconomic associations with illicit drug use, presenting a potential avenue for the use of this data to guide public health and healthcare policy decisions.

SARS-CoV-2 Infection Risk Factors among Maintenance Hemodialysis Patients and Health Care Personnel In Outpatient Hemodialysis Centers.

Increased risk of SARS-CoV-2 infection was associated with community prevalence.Increased risk of SARS-CoV-2 infection was associated with exposure to infected family members and personal infection prevention measures.

Clinical Performance of a Lateral Flow SARS-CoV-2 Total Antibody Assay.

BACKGROUND: Serological assays for SARS-CoV-2 are important tools for diagnosis in patients with negative RT-PCR testing, pediatric patients with multisystem inflammatory syndrome, and serosurveillance studies. However, lateral flow-based serological assays have previously demonstrated poor analytical and clinical performance, limiting their utility. METHODS: We assessed the ADEXUSDx COVID-19 lateral flow assay for agreement with diagnostic RT-PCR testing using 120 specimens from RT-PCR-positive patients, 77 specimens from symptomatic RT-PCR-negative patients, and 47 specimens obtained prepandemic. Specimens collected <14 days from symptom onset in RT-PCR-positive patients were compared relative to the Abbott SARS-CoV-2 IgG assay. RESULTS: The ADEXUSDx COVID-19 Test yielded an overall positive percent agreement (PPA) of 92.5% (95%CI 85.8 to 96.3) and negative percent agreement of 99.2% (95% CI 94.9-100.0) relative to RT-PCR and in prepandemic specimens. Relative to days from symptom onset, the PPA after 13 days was 100% (95% CI 94.2-100); from 7 to 13 days, 89.7 (95% CI 71.5-97.2); and from 0 to 7 days, 53.8 (95% CI 26.1-79.6). The overall agreement between the Abbott and ADEXUSDx assays was 80.9%. Twenty-five specimens were positive by both assays, 9 specimens were negative by both assays, and 8 specimens were positive by only the ADEXUSDx assay. CONCLUSIONS: We demonstrate high PPA and negative percent agreement of the ADEXUSDx COVID-19 assay and diagnostic testing by RT-PCR, with PPA approximately 90% by 7 days following symptom onset. The use of waived testing for antibodies to SARS-CoV-2 with high sensitivity and specificity provide a further tool for combatting the COVID-19 pandemic.

Cell specific peripheral immune responses predict survival in critical COVID-19 patients.

SARS-CoV-2 triggers a complex systemic immune response in circulating blood mononuclear cells. The relationship between immune cell activation of the peripheral compartment and survival in critical COVID-19 remains to be established. Here we use single-cell RNA sequencing and Cellular Indexing of Transcriptomes and Epitomes by sequence mapping to elucidate cell type specific transcriptional signatures that associate with and predict survival in critical COVID-19. Patients who survive infection display activation of antibody processing, early activation response, and cell cycle regulation pathways most prominent within B-, T-, and NK-cell subsets. We further leverage cell specific differential gene expression and machine learning to predict mortality using single cell transcriptomes. We identify interferon signaling and antigen presentation pathways within cDC2 cells, CD14 monocytes, and CD16 monocytes as predictors of mortality with 90% accuracy. Finally, we validate our findings in an independent transcriptomics dataset and provide a framework to elucidate mechanisms that promote survival in critically ill COVID-19 patients. Identifying prognostic indicators among critical COVID-19 patients holds tremendous value in risk stratification and clinical management.

Domain-specific biochemical and serological characterization of SARS-CoV-2 nucleocapsid protein.

Nucleocapsid proteins are essential for SARS-CoV-2 life cycle. Here, we describe protocols to gather domain-specific insights about essential properties of nucleocapsids. These assays include dynamic light scattering to characterize oligomerization, fluorescence polarization to quantify RNA binding, hydrogen-deuterium exchange mass spectrometry to map RNA binding regions, negative-stain electron microscopy to visualize oligomeric species, interferon reporter assay to evaluate interferon signaling modulation, and a serology assay to reveal insights for improved sensitivity and specificity. These assays are broadly applicable to RNA-encapsidated nucleocapsids. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).

Progression of SARS-CoV-2 Seroprevalence in St. Louis, Missouri, through January 2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seropositivity was assessed for 3,066 individuals visiting hospitals in St. Louis, Missouri, during July 2020, November 2020, or January 2021. Seropositivity in children increased from 5.22% in July to 21.16% in January. In the same time frame, seropositivity among adults increased from 4.52% to 19.03%, prior to initiation of mass vaccination. IMPORTANCE This study determined the percentage of children and adult samples from the St. Louis metropolitan area in Missouri with SARS-CoV-2 antibodies during three collection periods spanning July 2020 to January 2021. By January 2021, 20.68% of the tested individuals had antibodies. These results show the evolution of the SARS-CoV-2 pandemic in St. Louis, Missouri, and provide a snapshot of the extent of infection just prior to the start of mass vaccination.

HLA genetic polymorphism in patients with Coronavirus Disease 2019 in Midwestern United States.

The experience of individuals with Coronavirus Disease 2019 (COVID-19) ranges from asymptomatic to life threatening multi-organ dysfunction. Specific HLA alleles may affect the predisposition to severe COVID-19 because of their role in presenting viral peptides to launch the adaptive immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this population-based case-control study in the midwestern United States, we performed high-resolution HLA typing of 234 cases hospitalized for COVID-19 in the St. Louis metropolitan area and compared their HLA allele frequencies with those of 22,000 matched controls from the National Marrow Donor Program (NMDP). We identified two predisposing alleles, HLA-DRB1*08:02 in the Hispanic group (OR = 9.0, 95% confidence interval: 2.2-37.9; adjusted p = 0.03) and HLA-A*30:02 in younger African Americans with ages below the median (OR = 2.2, 1.4-3.6; adjusted p = 0.01), and several candidate alleles with potential associations with COVID-19 in African American, White, and Hispanic groups. We also detected risk-associated amino acid residues in the peptide binding grooves of some of these alleles, suggesting the presence of functional associations. These findings support the notion that specific HLA alleles may be protective or predisposing factors to COVID-19. Future consortium analysis of pooled cases and controls is warranted to validate and extend these findings, and correlation with viral peptide binding studies will provide additional evidence for the functional association between HLA alleles and COVID-19.

Characterization of SARS-CoV-2 nucleocapsid protein reveals multiple functional consequences of the C-terminal domain.

Nucleocapsid (N) encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays key roles in the replication cycle and is a critical serological marker. Here, we characterize essential biochemical properties of N and describe the utility of these insights in serological studies. We define N domains important for oligomerization and RNA binding and show that N oligomerization provides a high-affinity RNA-binding platform. We also map the RNA-binding interface, showing protection in the N-terminal domain and linker region. In addition, phosphorylation causes reduction of RNA binding and redistribution of N from liquid droplets to loose coils, showing how N-RNA accessibility and assembly may be regulated by phosphorylation. Finally, we find that the C-terminal domain of N is the most immunogenic, based on antibody binding to patient samples. Together, we provide a biochemical description of SARS-CoV-2 N and highlight the value of using N domains as highly specific and sensitive diagnostic markers.