Performance of High Throughput SARS-CoV-2 Antigen Testing Compared to Nucleic Acid Testing.

OBJECTIVE: Independent assessment of SARS-CoV-2 antigen (COV2Ag) tests remains important as varying performance between assays is common. We assessed the performance of a new high-throughput COV2Ag test compared to SARS-CoV-2 nucleic acid amplification tests (NAAT). METHODS: A total of 347 nasopharyngeal samples collected from January to October 2021 were assessed by NAAT as part of standard-of-care testing (CDC LDT or GeneXpert System, Cepheid) and COV2Ag using the ADVIA Centaur CoV2Ag assay (Siemens Healthineers). RESULTS: Among NAAT positive specimens we found 82.4% agreement and in NAAT negative specimens we found 97.3% agreement (overall agreement 85.6%). In symptomatic persons, COV2Ag agreed with NAAT 90.0% (n = 291), and in asymptomatic persons, 62.5% (n = 56). Agreement between positive NAAT and COV2Ag increased at lower cycle threshold (Ct) values. CONCLUSION: The COV2Ag assay exceeded the World Health Organization minimum performance requirements of ≥ 80% sensitivity and ≥ 97% specificity. The COV2Ag assay is helpful for large scale screening efforts due to high-throughput and reduced wait times.

Tixagevimab Plus Cilgavimab Does Not Affect the Interpretation of Electrophoretic and Free Light Chain Assays.

OBJECTIVES: There is concern that the anti-severe acute respiratory syndrome coronavirus 2 therapeutic monoclonal antibodies, used as preexposure prophylaxis in patients with multiple myeloma, may appear as a detectable monoclonal protein by electrophoretic methods, resulting in misinterpretation or inability to measure therapeutic responses in some patients. In this pilot study, we characterize the effect of tixagevimab plus cilgavimab (Evusheld; T + C) on interpretation of serum protein electrophoresis (SPE), immunofixation electrophoresis (IFE), and serum free light chain (sFLC) assays. METHODS: We performed spiking experiments with T + C at serum maximum concentration following a 300-mg dose (1× Cmax) and at 10 times the concentration of Cmax (10× Cmax) with pooled serum samples. SPE and IFE technical procedures were performed on the SPIFE 3000, and sFLC and immunoglobulin G1 (IgG1) subtype quantitation was performed on the Optilite. RESULTS: T + C-associated interference was not visible as an M-spike in normogammaglobulinemic pooled samples. Hypogammaglobulemic pooled samples at 10× Cmax demonstrated an M-spike in SPE and immunoglobulin Gκ pattern in IFE. No increases were noted in the results of sFLC or IgG1 levels. CONCLUSIONS: This study indicates that T + C at pharmacologic Cmax is unlikely to interfere with SPE, IFE, sFLC, or IgG1 analyses when spiked into patient serum samples, but further evaluation of recently injected patients may be warranted.

Characterization of Casirivimab Plus Imdevimab, Sotrovimab, and Bamlanivimab Plus Etesevimab-Derived Interference in Serum Protein Electrophoresis and Immunofixation Electrophoresis.

BACKGROUND: Therapeutic monoclonal antibodies can be a source of assay interference in clinical serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE), producing monoclonal bands that can be misinterpreted as a monoclonal gammopathy related to a B-cell or plasma cell neoplasm. The extent to which new anti-COVID-19 monoclonal antibodies produce this interference is unknown. METHODS: Casirivimab plus imdevimab, sotrovimab, and bamlanivimab plus etesevimab were spiked into patient serum samples to evaluate for SPEP/IFE interference, to characterize the position of therapy-derived bands relative to a reference band (either combined beta band or beta 1 band, depending on instrument platform), and to confirm heavy and light chain utilization of each medication. Serum samples from patients who had recently received casirivimab plus imdevimab or sotrovimab were also evaluated for comparison. RESULTS: When spiked into serum samples, all tested anti-COVID-19 monoclonal antibodies generated interference in SPEP/IFE. Importantly, the patterns of interference differed between spiked serum samples and serum from patients who had recently received casirivimab plus imdevimab or sotrovimab. CONCLUSIONS: Imdevimab can be added to the growing list of therapeutic monoclonal antibodies that produce sustained interference in SPEP/IFE. Although casirivimab and sotrovimab also produce assay interference in vitro, these antibodies are not reliably detected in serum from recently infused patients. The value of relative band position in recognizing bands that may represent therapeutic monoclonal antibodies is also emphasized. Clinicians and laboratorians should consider therapeutic monoclonal antibody interference in diagnostic SPEP/IFE and review a patient's medication list when new or transient monoclonal bands are identified.

Integrating High-Dose GLP-1 Receptor Agonists Into the Care of People With or at Risk for Type 2 Diabetes

According to the 2019 Centers for Disease Control and Prevention (CDC) Diabetes Report Card, the proportion of US adults living with diabetes has steadily increased since 1999, and in the last decade, the number of adults with diabetes has almost doubled. Excess weight contributes to insulin resistance and is the major factor driving the increasing prevalence of type 2 diabetes in the US. In fact, a recent study by researchers at Northwestern University School of Medicine, which examined National Health and Nutrition Survey and Multi-Ethnic Study of Atherosclerosis data from 2000 to 2017, estimates 30% to 53% of incident diabetes in the US can be attributed to obesity.Unfortunately, a high percentage of US adults are overweight and obese. Currently, the CDC estimates that 3 of every 4 US adults has a BMI greater than 25 kg/m2. People with obesity are more likely to develop metabolic conditions and are at an increased risk for serious illness from acute causes, such as coronavirus disease (COVID-19). Because of the high association of obesity with type 2 diabetes, efforts that address both conditions should be a priority for US health care providers. Until recently, highly effective drug therapy options were limited by narrow therapeutic windows and/or poor effectiveness.The purpose of this review is to provide an update on emerging data for high-dose glucagon-like peptide (GLP-1) receptor agonists to treat overweight or obesity in those at risk for or diagnosed with diabetes.

COVID-19 mRNA Vaccines May Cause False Reactivity in Some Serologic Laboratory Tests, Including Rapid Plasma Reagin Tests.

OBJECTIVES: Acute viral infections and some vaccines have been shown to increase false positivity in serologic assays. We assessed if the messenger RNA coronavirus disease 2019 (COVID-19) vaccines could cause false reactivity in common serologic assays in a pilot longitudinal cohort. METHODS: Thirty-eight participants with sera available prevaccination, 2 weeks after each vaccine dose, and monthly thereafter for up to 5 months were tested for common infectious disease serologies and antiphospholipid syndrome (APS) serology markers on the BioPlex 2200, Sure-Vue rapid plasma reagin (RPR), and Macro-Vue RPR. Twenty-two participants received the Moderna vaccine and 16 received the Pfizer vaccine. RESULTS: Most assays had no change in reactivity over the course of the sample draws, including APS markers. Epstein-Barr virus immunoglobulin G (IgG), measles IgG, and rubella immunoglobulin M all had possible false reactivity in one to two participants. RPR tests demonstrated false reactivity, with baseline nonreactive participant samples becoming reactive following vaccination. There were more false reactive participants (7/38) in the BioPlex RPR than in the Sure-Vue (2/38) and Macro-Vue (1/38) tests. All falsely reactive RPR tests were in participants who received the Moderna vaccine. CONCLUSIONS: Serologic assays with results that do not fit the clinical picture following COVID-19 vaccination should be repeated. Effects of false reactivity can last more than 5 months in some assays. In particular, RPR is susceptible to false reactivity, and there is variability among assays. Larger longitudinal studies are needed to determine the incidence and window of false reactivity.

Pediatric SARS-CoV-2 seroprevalence during mitigation procedures in Southwestern Pennsylvania.

Background: Children infected with SARS-CoV-2 are often asymptomatic or have only mild symptoms, leading to underestimation of disease prevalence in symptom-based testing strategies. Objectives: This study sought to determine pediatric SARS-CoV-2 disease burden during local mitigation efforts by using antibody testing to compare seroprevalence estimates to cumulative PCR prevalence estimates. Study design: In this cross-sectional study, we collected 1142 strict phase and 1196 relaxed phase remnant blood specimens from patients less than 19-years-old in southwestern Pennsylvania (SWPA). Patients were excluded if their residential zip code was outside the region of interest, if they were under 6-months-old, or they had recently received antibody-modifying treatments. Demographic, encounter, and laboratory electronic medical record information was extracted. Samples were tested for SARS-CoV-2 spike protein IgG using an EUA ELISA, and PCR results were recorded from county health department data. Seroprevalence and Clopper-Pearson exact 95% confidence intervals were calculated. Results: The observed seroprevalence of SARS-CoV-2 spike protein antibodies in children during strictest mitigation was 0.53% (95% CI 0.19, 1.14) and 0.92% (95% CI 0.46,1.64) during moderately relaxed. Strict and relaxed phase PCR-based prevalence were significantly higher, 2.87% (95% CI 1.95, 4.08) and 3.64 (95% CI 3.01, 4.38), respectively. Conclusions: Estimates of pediatric seroprevalence were significantly lower than cumulative PCR prevalence estimates, and less than adult seroprevalence estimates, potentially due to biological, population, or sampling differences. Biological differences in pediatric immune responses to SARS-CoV-2 may make serosurvey interpretation challenging and these differences warrant further study.

Multiplex assessment of SARS-CoV-2 antibodies improves assay sensitivity and correlation with neutralizing antibodies.

OBJECTIVES: Detection of antibodies to multiple SARS-CoV-2 antigens in a single assay could increase diagnostic accuracy, differentiate vaccination from natural disease, and aid in retrospective exposure determination. Correlation of binding antibody assessment in clinical assays with neutralizing antibodies is needed to better understand the humoral response to SARS-CoV-2 infection and establish of correlates of protection. METHODS: A cohort of 752 samples was used to assess specificity, sensitivity, and comparison to 6 other Conformitè Europëenne serologic assays for the BioRad SARS-CoV-2 IgG multiplex assay which measures receptor binding domain IgG (RBD), spike-S1 IgG (S1), spike-S2 IgG (S2), and nucleocapsid IgG (N). A subset of serial specimens from 14 patients was also tested for neutralizing antibodies (n = 61). RESULTS: Specificity for RBD and S1 IgG was 99.4% (n = 170) and 100% for S2 and N IgG (n = 170) in a cohort selected for probable interference. Overall assay concordance with other assays was >93% for IgG and total antibody assays and reached 100% sensitivity for clinical concordance at >14 days as a multiplex assay. RBD and S1 binding antibody positivity demonstrated 79-95% agreement with the presence of neutralizing antibodies. CONCLUSIONS: The BioRad SARS-CoV-2 IgG assay is comparable to existing assays, and achieved 100% sensitivity when all markers were included. The ability to measure antibodies against spike and nucleocapsid proteins simultaneously may be advantageous for complex clinical presentations, epidemiologic research, and in decisions regarding infection prevention strategies. Additional independent validations are needed to further determine binding antibody and neutralizing antibody correlations.

Differential Antibody Response to mRNA COVID-19 Vaccines in Healthy Subjects.

Knowledge about development and duration of virus-specific antibodies after COVID-19 vaccination is important for understanding how to limit the pandemic via vaccination in different populations and societies. However, the clinical utility of postvaccination testing of antibody response and selection of targeted SARS-CoV-2 antigen(s) has not been established. The results of such testing from clinical teams independent from vaccine manufacturers are also limited. Here, we report the initial results of an ongoing clinical study on evaluation of antibody response to four different SARS-CoV-2 antigens after first and second dose of Pfizer and Moderna mRNA vaccines and at later time points. We revealed a peak of antibody induction after the vaccine boosting dose with a gradual decline of antibody levels at later time. Anti-nucleocapsid antibody was not induced by spike protein-encoding vaccines and this may continue to serve as a marker of previous SARS-CoV-2 infection. No differences between the two vaccines in terms of antibody response were revealed. Age and gender dependencies were determined to be minimal within the healthy adult (but not aged) population. Our results suggest that postvaccination testing of antibody response is an important and feasible tool for following people after vaccination and selecting individuals who might require a third dose of vaccine at an earlier time point or persons who may not need a second dose due to previous SARS-CoV-2 infection. IMPORTANCE Now that authorized vaccines for COVID-19 have been widely used, it is important to understand how they induce antivirus antibodies, which antigens are targeted, how long antibodies circulate, and how personal health conditions and age may affect this humoral immunity. Here, we report induction and time course of multiple anti-SARS-CoV-2 antibody responses in healthy individuals immunized with Pfizer and Moderna mRNA vaccines. We also determined the age and gender dependence of the antibody response and compared antibody levels to responses seen in those who have recovered from COVID-19. Our results suggest the importance of screening for antibody response to multiple antigens after vaccination in order to reveal individuals who require early and late additional boosting and those who may not need second dose due to prior SARS-CoV-2 infection.

A Cross-Sectional Study of SARS-CoV-2 Seroprevalence between Fall 2020 and February 2021 in Allegheny County, Western Pennsylvania, USA.

Seroprevalence studies are important for understanding the dynamics of local virus transmission and evaluating community immunity. To assess the seroprevalence for SARS-CoV-2 in Allegheny County, an urban/suburban county in Western PA, 393 human blood samples collected in Fall 2020 and February 2021 were examined for spike protein receptor-binding domain (RBD) and nucleocapsid protein (N) antibodies. All RBD-positive samples were evaluated for virus-specific neutralization activity. Our results showed a seroprevalence of 5.5% by RBD ELISA, 4.5% by N ELISA, and 2.5% for both in Fall 2020, which increased to 24.7% by RBD ELISA, 14.9% by N ELISA, and 12.9% for both in February 2021. Neutralization titer was significantly correlated with RBD titer but not with N titer. Using these two assays, we were able to distinguish infected from vaccinated individuals. In the February cohort, higher median income and white race were associated with serological findings consistent with vaccination. This study demonstrates a 4.5-fold increase in SARS-CoV-2 seroprevalence from Fall 2020 to February 2021 in Allegheny County, PA, due to increased incidence of both natural disease and vaccination. Future seroprevalence studies will need to include the effect of vaccination on assay results and incorporate non-vaccine antigens in serological assessments.

Unexpected False-Positive Rates in Pediatric SARS-CoV-2 Serology Using the EUROIMMUN Anti-SARS-CoV-2 ELISA IgG Assay.

OBJECTIVES: Serologic assay performance studies for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-​2) in pediatric populations are lacking, and few seroprevalence studies have routinely incorporated orthogonal testing to improve accuracy. METHODS: Remnant serum samples for routine bloodwork from 2,338 pediatric patients at UPMC Children's Hospital of Pittsburgh were assessed using the EUROIMMUN Anti-SARS-CoV-2 ELISA IgG (EuroIGG) assay. Reactive cases with sufficient volume were also tested using 3 additional commercial assays. RESULTS: Eighty-five specimens were reactive according to the EuroIGG, yielding 3.64% (95% confidence interval [CI], 2.91%-4.48%) seropositivity, of which 73 specimens had sufficient remaining volume for confirmation by orthogonal testing. Overall, 19.18% (95% CI, 10.18%-28.18%) of samples were positive on a second and/or third orthogonal assay. This 80.82% false positivity rate is disproportionate to the expected false positivity rate of 50% given our pediatric population prevalence and assay performance. CONCLUSIONS: In pediatric populations, false-positive SARS-CoV-2 serology may be more common than assay and prevalence parameters would predict, and further studies are needed to establish the performance of SARS-CoV-2 serology in children.

Rapid Detection of SARS-CoV-2 Antigens Using High-Purity Semiconducting Single-Walled Carbon Nanotube-Based Field-Effect Transistors.

Early diagnosis of SARS-CoV-2 infection is critical for facilitating proper containment procedures, and a rapid, sensitive antigen assay is a critical step in curbing the pandemic. In this work, we report the use of a high-purity semiconducting (sc) single-walled carbon nanotube (SWCNT)-based field-effect transistor (FET) decorated with specific binding chemistry to assess the presence of SARS-CoV-2 antigens in clinical nasopharyngeal samples. Our SWCNT FET sensors, with functionalization of the anti-SARS-CoV-2 spike protein antibody (SAb) and anti-nucleocapsid protein antibody, detected the S antigen (SAg) and N antigen (NAg), reaching a limit of detection of 0.55 fg/mL for SAg and 0.016 fg/mL for NAg in calibration samples. SAb-functionalized FET sensors also exhibited good sensing performance in discriminating positive and negative clinical samples, indicating a proof of principle for use as a rapid COVID-19 antigen diagnostic tool with high analytical sensitivity and specificity at low cost.

SARS-CoV-2 Serologic Immune Response in Exogenously Immunosuppressed Patients.

BACKGROUND: While it is presumed that immunosuppressed patients, such as solid organ transplant recipients on immunosuppression, are at greater risk from SARS-CoV-2 infection than the general population, the antibody response to infection in this patient population has not been studied. METHODS: In this report, we follow the anti-SARS-CoV-2 antibody levels in patients with COVID-19 who are undergoing exogenous immunosuppression. Specifically, we studied the antibody response of 3 solid organ transplant recipient patients, 3 patients who take daily inhaled fluticasone, and a patient on etanercept and daily inhaled fluticasone, and compared them to 5 patients not on exogenous immunosuppression. RESULTS: We found that the solid organ transplant patients on full immunosuppression are at risk of having a delayed antibody response and poor outcome. We did not find evidence that inhaled steroids or etanercept predispose patients to delayed immune response to SARS-CoV-2. CONCLUSION: The data presented here suggest that solid organ transplant recipients may be good candidates for early targeted intervention against SARS-CoV-2.

Variable Performance in 6 Commercial SARS-CoV-2 Antibody Assays May Affect Convalescent Plasma and Seroprevalence Screening.

OBJECTIVES: Serologic detection of prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is needed for definition of convalescent plasma donors, for confounding SARS-CoV-2 presentation, and for seroprevalence studies. Reliable serologic assays with independent validation are required. METHODS: Six SARS-CoV-2 antibody assays from Beckman Coulter, Euroimmun (IgG, IgA), Roche, and Siemens (Centaur, Vista) were assessed for specificity (n = 184), sensitivity (n = 154), and seroconversion in a defined cohort with clinical correlates and molecular SARS-CoV-2 results. RESULTS: Assay specificity was 99% or greater for all assays except the Euroimmun IgA (95%). Sensitivity at more than 21 days from symptom onset was 84%, 95%, 72%, 98%, 67%, and 96% for Beckman Coulter, Centaur, Vista, Roche, Euroimmun IgA, and Euroimmun IgG, respectively. Average day of seroconversion was similar between assays (8-10 d), with 2 patients not producing nucleocapsid antibodies during hospitalization. CONCLUSIONS: SARS-CoV-2 nucleocapsid antibodies may be less reliably produced early in disease than spike protein antibodies. Assessment of convalescent plasma donors at more than 30 days from symptom onset and seroprevalence studies should use assays with defined sensitivity at time points of interest because not all assays detected antibodies reliably at more than 30 days.

Immunocompromised Seroprevalence and Course of Illness of SARS-CoV-2 in One Pediatric Quaternary Care Center.

BACKGROUND: The burden of coronavirus disease 2019 (COVID-19) is poorly understood in pediatric patients due to frequent asymptomatic and mild presentations. Additionally, the disease prevalence in pediatric immunocompromised patients remains unknown. METHODS: This cross-sectional study tested convenience samples from pediatric patients who had clinically indicated lab work collected and an immunocompromising condition, including oncologic diagnoses, solid organ transplant (SOT), bone marrow transplant, primary immunodeficiency, and rheumatologic conditions or inflammatory bowel disease on systemic immunosuppression, for the presence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RESULTS: We tested sera from 485 children and observed SARS-CoV-2 seroprevalence of 1.0% (Confidence Interval [CI] 95%: 0.3%-2.4%). Two patients were positive by nasopharyngeal (NP) swab Reverse transcriptase polymerase chain reaction (RT-PCR), but only 1 seroconverted. Patients with oncologic diagnoses or SOT were most likely to be tested for COVID-19 when presenting with respiratory illness as compared with other groups. CONCLUSIONS: Seroprevalence of antibodies to SARS-CoV-2 in immunocompromised children was similar to that of an immunocompetent pediatric population (0.6%, CI 95%: 0.3%-1.1%), suggesting an adequate antibody response. However, none of the patients who tested positive for antibodies or via NP RT-PCR had more than a mild illness course and 2 patients did not have any reported illness, suggesting that SARS-CoV-2 may not cause a worse clinical outcome in immunosuppressed children, in contrast to immunocompromised adults.

Evaluation of SARS-CoV-2 prototype serologic test in hospitalized patients.

OBJECTIVES: Humoral immune response to SARS-CoV-2 infection has been reported in several patient cohorts with results that vary by method and population studied due to the lack of reliable commercial assays available as the pandemic initially spread. We sought to clinically assess commercial prototype SARS-CoV-2 IgG and IgA assays for use in screening for prior infection and convalescent plasma donation. DESIGN AND METHODS: Prototype SARS-CoV-2 IgG and IgA assays from Euroimmun were assessed utilizing remnant specimens. Specificity testing used specimens in their convalescent window for the common coronaviruses and other infectious diseases known to be associated with increased non-specificity in serologic assays. Sensitivity testing utilized serial specimens from molecularly confirmed SARS-CoV-2 critically ill patients to assess seroconversion. Utilizing recombinant spike protein we also developed a competitive confirmation procedure to increase assay specificity. RESULTS: We determined specificity to be 97% and 81%, respectively, when indeterminate samples were considered positive and 99% and 86% when indeterminate samples were considered negative. We developed a new confirmation methodology to enhance the specificity of the assays with an anticipated specificity of 98% for IgA. Valuation of hospitalized COVID-19 patients determined median IgA seroconversion to be 8 days and IgG 10 days. Neither level nor timing of antibody response correlated with days on ventilation. End titer measurements indicate that validated improved assays may be capable of semi-quantitative measurement. CONCLUSIONS: We found these assays to be clinically acceptable for the high prevalence population tested, for instance, for convalescent plasma donation.

SARS-CoV-2 growth, furin-cleavage-site adaptation and neutralization using serum from acutely infected hospitalized COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1 : P4 and Munich : P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1 : P6 and minor variants in the Munich : P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.