key: cord-0989944-zovj2vt5 authors: Sarina Yang, He; Racine-Brzostek, Sabrina E.; Lee, William T.; Hunt, Danielle; Yee, Jim; Chen, Zhengming; Kubiak, Jeffrey; Cantu, Miguel; Hatem, Layla; Zhong, Elaine; D'Ambrosio, Danielle; Chadburn, Amy; Westblade, Lars; Glesby, Marshall; Loda, Massimo; Cushing, Melissa M.; Zhao, Zhen title: SARS-CoV-2 antibody characterization in emergency department, hospitalized and convalescent patients by two semi-quantitative immunoassays date: 2020-06-04 journal: Clin Chim Acta DOI: 10.1016/j.cca.2020.06.004 sha: 552b8a124b64ea4f18541ef23367369f43ff08d2 doc_id: 989944 cord_uid: zovj2vt5 BACKGROUND: In the ongoing COVID-19 pandemic, there is an urgent need for comprehensive performance evaluation and clinical utility assessment of serological assays to understand the immune response to SARS-CoV-2. METHODS: IgM/IgG and total antibodies against SARS-CoV-2 were measured by a cyclic enhanced fluorescence assay (CEFA) and a microsphere immunoassay (MIA), respectively. Independent performance evaluation included imprecision, reproducibility, specificity and cross-reactivity (CEFA n=320, MIA n=364). Clinical utility was evaluated by both methods in 87 patients at initial emergency department visit, 28 during subsequent hospitalizations (106 serial samples), and 145 convalescent patients. Totally 916 patients and 994 samples were evaluated. RESULTS: Agreement of CEFA and MIA was 90.4%-94.5% (Kappa: 0.81-0.89) in 302 samples. CEFA and MIA detected SARS-CoV-2 antibodies in 26.2% and 26.3%, respectively, of ED patients. Detection rates increased over time reaching 100% after 21 days post-symptom onset. Longitudinal antibody kinetic changes by CEFA and MIA measurements correlated well and exhibited three types of seroconversion. Convalescent sera showed a wide range of antibody levels. CONCLUSION: Rigorously validated CEFA and MIA assays are reliable for detecting antibodies to SARS-CoV-2 and show promising clinical utility when evaluating immune response in hospitalized and convalescent patients, but are not useful for early screening at patient’s initial ED visit. The ongoing global pandemic of Coronavirus Disease-2019 has rapidly spread with globally over 3.7 million confirmed cases and over 259,000 total deaths as of May 5, 2020 [1] . Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2), the cause of COVID-19, is highly contagious and can result in significant mortality among susceptible individuals with comorbidities. Acute symptoms and signs of SARS-CoV-2 infection are highly nonspecific and include fever, cough, fatigue, myalgia, and dyspnea with some patients progressing to pneumonia [2] [3] [4] . However some other individuals are asymptomatic carriers [5] [6] [7] . These characteristics of the disease create an urgent need to develop serological tests to identify asymptomatic silent infections, evaluate patient immune response, better predict disease progression and improve our understanding of the epidemiology, including transmission patterns, of SARS-CoV-2. Serological testing could also play an important role for de-isolation procedures [8] and implementation of convalescent plasma therapy for ill COVID-19 patients [9, 10] . Throughout the COVID pandemic, a wide variety of serological tests have entered the global market, including, but not limited to, colloidal gold immunochromatographic assays, magnetic chemiluminescent immunoassays, enzyme-linked immunosorbent assays (ELISA), and rapid test cassettes and dipsticks [4, 11, 12] . Due to the growing public health emergency and in an effort to facilitate rapid expansion of testing capacity, the United States Food and Drug Administration (FDA) issued a policy on mid-March 2020 [13] and a revised policy in early May [14] , allowing for the development of COVID-19 diagnostic testing in the clinical health care and commercial settings through the Emergency Use Authorization (EUA) program. Over 100 manufacturers have notified the FDA that they are offering or plan to offer serological tests in the United States, but as yet only 12 assays have received EUA clearance [15] . Furthermore, there has been a lack of rigorous validation and performance evaluation of the available serological assays in COVID-19 negative and positive populations as well as a lack of thorough comparison between different serological testing platforms. Such data are urgently needed to evaluate the clinical utility and also the limitations of serological tests, as there has been significant controversy over the diagnostic and prognostic value of antibody testing. In addition, the potential role of serological antibody testing in epidemiological studies and in the accurate identification of convalescent plasma donors for COVID-19 patients is not known. This study was approved by the Institutional Review Board (#20-03021671) of Weill Cornell Medicine (site 1). The testing at Wadsworth Center at the New York State Department of Health (NYS DOH) (site 2) is waived for public health purposes. Different cohorts of patient serum samples were included in this study for evaluating analytical and clinical performance of the two assays. A chart of patients and samples used in this study is shown in Figure 1 . Serum specimens (n=320), collected from the pre-COVID 19 ED patients in July 2019, were tested to validate the specificity of the CEFA assay. Serum from 256 pre-COVID-19 healthy blood donors collected before 2019 were used to validate the specificity of the MIA assay. Thirty sera from patients treated for recent non-COVID- 19 The convalescent serum samples were from 145 patients who tested positive by SARS-CoV-2 RT-PCR or had a COVID-19-like illness but had not undergone SARS-CoV-2 RT-PCR testing. Based on patients' self-description, they had been symptom free for at least 14 days. The IgM and IgG antibodies against SARS-CoV-2 in serum were measured using the Pylon This assay has received US FDA EUA clearance. Specimens were assessed for the presence of total antibody using the SARS-CoV-2 MIA. Antigen for the MIA method is the recombinant SARS-CoV-2 NP. Analysis was performed using a Luminex 100 Analyzer (Luminex Corporation, Austin, TX. [Additional methodology described in supplemental material]. Positive and negative patient samples are run on a daily basis when patient samples are tested. The cut off values of both methods were determined by mean of non-COVID-19 samples plus 6 Standard Deviation (SD). The index value (IV) was determined by the instrument readout of the test sample divided by instrument readout at cut off. An indeterminate IV was mean (of IgG for CEFA and total antibodies for MIA, respectively) plus 3SD divided by the instrument readout at cut off. Samples with an IV ≥ 1 and 1.78 were designated as positive for CEFA and MIA, respectively. For the CEFA method, high and low levels of IgM and IgG QC, respectively, were run on 20 days. Mean, SD, CV were calculated. Six patient samples with negative, indeterminate, and positive (low, medium and high levels) IgM and IgG were run on five consecutive days. For the MIA method, mean, SD, CV were calculated for positive and negative QC which were run on 30 days. SARS-CoV-2 RT-PCR assay was performed using the RealStar SARS-CoV-2 RT-PCR kit 1.0 (Altona Diagnostics USA, Inc., Plain City, OH), which qualitatively detects SARS-CoV-2 RNA extracted from nasopharyngeal swab specimens. Respiratory Pathogen 2 PCR (RPP) was performed using the FilmArray Respiratory Pathogen 2 Panel (BioFire Diagnostics, LLC, Salt Lake City, UT) for the simultaneous qualitative detection and identification of multiple respiratory viruses and bacteria. [Additional information provided in supplemental material]. Data are presented as mean ± SD or median with interquartile range ( The plot of index values (log10 scale) and a simulated trending curve of IgM/IgG and total antibody are shown in Figures 2 a and b , respectively. Overall, IgM (CEFA, Figure 2a) and IgG seroconversion earlier than that of IgM (5 patients). Five patients, discharged less than 14 days after admission, did not have detectable antibody levels. Thus, it is unclear if these patients had sufficient time to develop an antibody response. Further analysis suggests that changes in IgM, IgG and total antibody levels over time are not significantly different between the ventilator and non-ventilator groups in our dataset (p = 0.30 for IgM, p = 0.84 for IgG, p = 0.1 for total antibody). Figure 4a and b, respectively. This study serves as an important example of a collaborative effort between an academic clinical laboratory and state government agency. This study sheds light on the performance and potential clinical utility of two SARS-CoV-2 antibody testing methodologies during the COVID-19 outbreak in New York City. Overall, both CEFA and MIA assays demonstrated excellent analytical performance in their independent validations as well as concordance in ED, inpatient and convalescent samples. While the result reporting differs between the two assays, where one detects IgM and IgG separately and the other targets total antibody, we found that the detection rate at any given time period postsymptom onset did not differ significantly, with both assays demonstrating sensitivities ≤ 25% when tested <7 days after symptoms began, reaching 100% > 21 days after symptom onset. These results are consistent with previous studies [17] evaluating recombinant immunofluorescence or chemiluminescent immunoassays [18, 19] . In contrast, RT-PCR testing demonstrates its highest level of sensitivity during the first week of symptoms, and then gradually declines in the next few weeks [20, 21] . We demonstrate that serological testing has limited diagnostic value in screening symptomatic patients during their first ED visit. As reporting of symptom onset in ED patients is subject to recall bias, a variation in time to antibody detection was observed, with most RT-PCR Prior studies reported most individuals infected with SARS-CoV-1 mounted an antibody response [23] [24] [25] whereas MERS-CoV infected patients with mild or asymptomatic infections exhibited varied immune responses, which at times were undetectable by antibody assays [26, 27] . While there is growing data on the antibody response to SARS-CoV-2 infection [18, 28, 29] , the level and duration of the anti-SARS-CoV-2 antibodies in asymptomatic and mildly symptomatic COVID-19 patients is still uncertain. In the present dataset, all 18 patients in the mechanically ventilated group seroconverted. This is in contrast to 5 (CEFA) and 4 (MIA) out of 10 patients in the non-ventilated group who had undetectable IgM and IgG levels during their hospital stay. Four patients were discharged < 10 days post-symptom onset and 1 patient had undetectable antibodies when discharged on day 13 after symptom onset. Thus, this retrospective study cannot provide additional data on these patients. However, the perceived suboptimal antibody responses is likely due to short hospitalizations resulting in the lack of specimen for analysis during the intermediate and late stages of the disease. More specimens from mildly ill patients will need to be collected for longer time periods in order to better understand patterns of SARS-CoV-2 immune response. This information is essential to discussions regarding reentry into the workforce by asymptomatic individuals post-infection. It has been suggested that worse outcomes and increased disease severity with COVID-19 may correspond with increased SARS-CoV-2 IgG levels or a higher titer of SARS-CoV-2 antibodies, when compared to those with less severe disease [20, 30, 31] . Although the current study found that the positivity rate of antibodies increased with time after symptom onset for both methods, there was no statistical difference in antibody levels based on ventilation status. Assay variation may explain the lack of correlation between antibody levels and severity of disease, as our methods currently are validated only for qualitative SARS-CoV-2 antibody analysis and its semiquantitative use may not give the analytical resolution needed for such studies. Bias may also exist in that patients with milder symptoms did not present to the hospital and thus, there is no comparison between this missing cohort and the cohort of hospitalized patients which displayed more severe symptoms. Functional assays, such as neutralizing antibody assays, have been proposed to evaluate the efficacy of vaccinations and due to the complicated interplay between virus and the immunologic host response [32] , neutralizing antibody assays [30] may be better suited for such correlation studies. Furthermore, this study did not extensively follow longitudinally patient antibody levels and consequently, the study was likely underpowered in its ability to see this secondary outcome measure. Additional independent large-cohort studies would be needed to further study these previously described findings. Both the FDA and CDC stress the importance of serological testing for the detection of prior infection in asymptomatic individuals and those presenting late in illness as well as identifying individuals who have mounted an immune response to SARS-CoV-2 [33, 34] . Our data suggest that there is a wide range of antibody levels in convalescent serum. Of note, the distribution of antibody positivity in convalescent sera examined in this study cannot be used to determine antibody prevalence and levels in the general population, as these were random samples from patients recovered from COVID-19 and COVID-19-like illnesses. Cases have been reported that convalescent plasma therapy improved the clinical outcomes by neutralizing viremia in severe COVID-19 cases [10, 35] . Further studies are needed to investigate whether IgG or total antibody levels correlate with neutralizing antibody levels, thereby identifying potential therapeutic serum donors. In summary, we performed a thorough analytical validation and clinical evaluation of two semi-quantitative SARS-CoV-2 immunoassays. Our results demonstrated the clinical utilities of serological testing in evaluating inpatients and convalescent patients. The sensitivity of the current immunoassays are not suitable for early-stage screening. Future studies are needed to investigate the immune response in asymptomatic and mildly ill patient population, and to understand the correlation between total antibody and neutralizing antibody levels. Conflict of Interest: ZZ received seed instruments and sponsored travel from ET Healthcare. The manufacturers did not review the article and had no input on data analysis prior to the manuscript submission. An interactive web-based dashboard to track COVID-19 in real time A new coronavirus associated with human respiratory disease in China Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study A Peptide-based Magnetic Chemiluminescence Enzyme Immunoassay for Serological Diagnosis of Corona Virus Disease 2019 (COVID-19) COVID-19 transmission through asymptomatic carriers is a challenge to containment. Influenza Other Respir Viruses Detection of Novel Coronavirus by RT-PCR in Stool Specimen from Asymptomatic Child Clinical characteristics of 3,062 COVID-19 patients: a meta-analysis Diagnostic Testing for Severe Acute Respiratory Syndrome-Related Coronavirus-2: A Narrative Review Deployment of convalescent plasma for the prevention and treatment of COVID-19 Effectiveness of convalescent plasma therapy in severe COVID-19 patients Evaluation of Enzyme-Linked Immunoassay and Colloidal Gold-Immunochromatographic Assay Kit for Detection of Novel Coronavirus (SARS-Cov-2) Causing an Outbreak of Pneumonia Food and Drug Administration. Policy for Diagnostic Tests for Coronavirus Disease-2019 during the Public Health Emergency Policy for Coronavirus Disease-2019 Tests During the Public Health Emergency, 2020. 15. Administration FaD. Emergency Use Authorizations Sex-specific 99th percentile upper reference limits for high sensitivity cardiac troponin assays derived using a universal sample bank Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19) Virological assessment of hospitalized patients with COVID-2019 Diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019 Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 Interpreting Diagnostic Tests for SARS-CoV-2 Antibody responses to SARS-CoV-2 in patients with COVID-19 Immunofluorescence assay for serologic diagnosis of SARS Longitudinal analysis of Severe Acute Respiratory Syndrome (SARS) coronavirus-specific antibody in SARS patients Characterization of a novel coronavirus associated with severe acute respiratory syndrome Sensitive and Specific Detection of Low-Level Antibody Responses in Mild Middle East Respiratory Syndrome Coronavirus Infections Comparative Serological Study for the Prevalence of Anti-MERS Coronavirus Antibodies in High-and Low-Risk Groups in Qatar Serological and molecular findings during SARS-CoV-2 infection: the first case study in Finland Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications Immune phenotyping based on neutrophilto-lymphocyte ratio and IgG predicts disease severity and outcome for patients with Utilization of serologic assays to support efficacy of vaccines in nonclinical and clinical trials: meeting at the crossroads Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma Total 42 SARS2-CoV RT-PCR positive patients, 28 of them had serial samples. Total 120 samples for CEFA and 114 samples for MIA Comprehensive evaluation of 2 SARS-CoV-2 antibody assays with excellent agreement. 2. Rigorously validated antibody tests are reliable to detect antibody kinetic change Three types of seroconversion were observed in hospitalized COVID-19 patients. 4. Convalescent sera show a wide range of antibody levels. 5. Current antibody testing is not useful in early screening for COVID-19 CRediT author statement He Sarina Yang: Conceptualization, methodology, validation, Investigation, writing, review, and editing, data curation, visualization; Sabrina E. Racine-Brzostek: conceptualization, methodology, Investigation, writing, editing, data curation. William T Lee and Danielle Hunt: validation, analysis Jim Yee: investigation, software; Zhengming Chen: formal analysis Amy Chadburn: writing and editing; Lars Westblade: investigation Cushing: supervision, editing; Zhen Zhao: : Conceptualization, methodology, validation, Investigation, writing, review, and editing, data curation, visualization, supervision The authors thank the contribution of the following scientists from Wadsworth Center, Diagnostic Immunology Laboratory New York State Department of Health: K. McDonough, K. Kulas, R. Bievenue, S. Bush, K. Carson, V. Demarest, A. Furuya, K. Howard, M. Marchewka, R. Stone. We thank Dr. Fred Apple from University of Minnesota and Dr Alan Wu from University of California San Francisco for their help with manuscript review. JY collected the specimens and performed the experiments. MC, LH, JK, EZ and DD collected data. WL and DH analyzed the data, performed experiments, and edited the manuscript. SY analyzed the data, generated figures and wrote the manuscript. SRB analyzed the data and edited the manuscript, ZZ analyzed the data, performed experiments, edited the manuscript and supervised this study. ZC analyzed the data and edited the manuscript. AC, LW and MG edited the manuscript. MC and ML supervised this study and edited the manuscript.