ABSTRACT
Background: Sero-studies of SARS-CoV-2 have used antibody (Ab) responses to spike (S) and nucleocapsid (N) antigens to differentiate mRNA vaccinated (S+/N-) from infected (S+/N+) individuals. We performed testing on wellcharacterized subjects to determine how repeated vaccination or infection, and time from those exposures, influence these Ab levels. Method(s): Samples from individuals with known infection status: prepandemic negative controls n=462;first-time infected n=237 (~45 days post);vaccinated after infection n= 34 (~40 days post-vaccination and ~180 days post-infection);fully vaccinated n=158 (~50 days post);boosted n=31 (~30 days post);breakthrough n=18 (~14 days post-infection);reinfected n=10 (varied). Longitudinal samples (n=51) from subjects with evidence of reinfection (symptoms and/or positive rapid antigen test), were tested to determine the impact of the order of infection and/or vaccination on the magnitude of the anti-S and anti-N IgG Ab detected in the blood. Testing was performed with MesoScale Diagnostics (Gaithersburg, MD) assay. Outcomes are presented in WHO International Binding Antibody Units (BAU/mL). The cutoff for a positive result was 18 BAU for S and 12 BAU for N. Result(s): The median amount of Ab (IQR) in BAU for each group (Figure A) was: pre-pandemic negative controls S 0.53(0.27,1.03), N 0.55(0.18,1.67);first-time infected S 114(51,328), N 70(29,229);vaccinated after infection S 4367(2479,4837), N 15(7,35);fully vaccinated S 998(586,1529), N 0.31(0.16,0.68);boosted S 2988(1768,3522), N 0.59(0.32,1.03);breakthrough S 2429(2032,3413), N 2.5(0.93,8.6);reinfected S 1533(486,4643), N 7.8(2.6,62). For the breakthrough and second infections 17% and 40% were seropositive to N, respectively. Longitudinal analysis (Figure B) of those with multiple infections showed that all those with a positive rapid antigen test for their second infection had an increase in N Ab. Conclusion(s): The prevalence of antibodies to nucleocapsid cannot be used to determine the proportion of individuals infected to SARS-CoV-2 in a vaccinated population. Booster, repeated, and breakthrough infections are associated with IgG Ab levels to S >400 BAU/mL. A majority of breakthrough infections did not elicit an Ab response to N. For those with repeated infection, a minority elicited antibody responses to N. This could be related to misdiagnosis or the burden of infection, as only those who were positive by rapid antigen assay (indicative of a high viral load) had an increase in N Ab.
ABSTRACT
Background: The prevalence of vaccinated, previously infected, and individuals at risk of SARS-CoV-2 infection is important for epidemiologic studies and public health interventions. Asymptomatic infections and reluctance to disclose vaccination status hinder accurate assessments of the current state of the epidemic. Since COVID-19 vaccines generate immune responses to spike (S1), but not nucleocapsid (N), it is possible to differentiate between vaccinated, infected, and unexposed individuals by comparing antibody reactivity to each antigen. The MSD V-Plex SARS-CoV-2 IgG assay can potentially differentiate each state in one test by simultaneously evaluating IgG reactivity to the S1, receptor binding domain (RBD), and N proteins. Methods: The MSD assay was validated with three sample sets: known vaccination with no previous infection (n=158);known infected and not vaccinated (n=157);and samples collected prior to the COVID-19 pandemic in 2016 (n=144). Of the previously infected individuals, 15 (9.6%) were hospitalized;sample collection occurred a median of 48 days after a PCR-positive result. Using an algorithm, samples with positive results on both S1 and RBD but negative on N were classified as vaccinated. Samples with a positive result on all three proteins were considered to be infected with the possibility of subsequent vaccination. Any other result was classified as unexposed. Sensitivity and specificity for each state were calculated. Results: Reactivity to each antigen is shown in the figure. 100% (95% confidence interval [CI] 97.7-100), 92% (95% CI 86.3-95.5), and 0.7% (95% CI 0.02-3.8) of vaccinated, infected, and unexposed samples were positive for S1. 100% (95% CI 97.7-100.0%), 91% (95% CI 85.5-95.0%), and 0.7% (95% CI 0.02-3.8%) of vaccinated, infected and unexposed samples were positive for RBD. 0% (95% CI 0-2.3), 86% (95% CI 79.6-91.0), and 2.1% (95% CI 0.4-6.0) of vaccinated, infected and unexposed samples were positive for N. Algorithm sensitivity and specificity for classification of vaccinated samples were 100% (95% CI 97.7-100) and 96.7 (95% CI 94-98.4). For the classification of samples from previously infected individuals, sensitivity and specificity were 83.4% (95% CI 76.7-88.9) and 100% (95% CI 98.8-100). Conclusion: This study establishes the sensitivity and specificity for a high-throughput assay ideal for SARS-CoV-2 seroprevalence studies. Future research should focus on applying this assay in health care settings to guide practice and policy to mitigate the pandemic.
ABSTRACT
Background: Live virus micro-neutralization (MN) is the gold standard for quantifying the neutralizing titer (NT) of antibodies to SARS-CoV-2. However, performing MN is labor intensive and requires a biosafety level 3 laboratory. We assessed the performance of 8 immunoassays which measure SARS-CoV-2 NT and compared them to gold standard MN results. Methods: Samples from 269 individuals known to previously be SARS-CoV-2 PCR+ (i.e., convalescent individuals, <10% hospitalized) and 200 pre-pandemic individuals were evaluated on 3 lateral flow immunoassays (LFAs;Wondfo Colloidal Gold, Wondfo Colored Microsphere, Wondfo Finecare) and 5 enzyme-linked immunoassays (ELISAs;ImmunoRank, GenScript, Cusabio, Euroimmun NeutraLISA, Euroimmun QuantiVac). MN was performed on all samples from convalescent individuals;results were classified as undetectable vs any detection of MN NT (NT<20 vs. NT>20), as well as high and low MN NT (NT>80 vs. NT<80). Receiver operating curve analysis was used to assess accuracy for detecting levels of NT. The area under the curve (AUC) was calculated for the manufacturer's cut off and empirically to identify the best discriminatory cut off value. Cohen's kappa statistics were calculated to assess categorical agreement and Spearman's rank statistics were calculated to assess correlations. Results: Of the 269 convalescent plasma samples, 89 (33%) had MN NT values <20 (undetectable) and 117 (43%) >80 (high NT). Using the manufacturer's cutoffs, sensitivity for detection of samples with any NT ranged from 79% to 100%, and the false-positive rate (ie, classifying samples with undetectable NT as positive) was highest for LFAs (72% to 84%) and ranged from 14% to 69% for the ELISAs. For all assays except the ImmunoRank and NeutraLISA ELISAs, discrimination to identify samples with any NT was improved by raising the cut off values (Table). AUCs of ∼0.94 to discriminate high NT samples could be achieved for all quantifiable assays using an adjusted cut off value. Cohen's kappa statistic ranged from 0.20 to 0.69. Spearman's rank correlation between each assay and NT value ranged from 0.73 to 0.86. Using the manufacturer's cutoffs, specificity on pre-pandemic samples was ≥98% for all assays except for Cusabio which was 86%. Conclusion: The performance of immunoassays using manufacturer's cutoff to discriminate samples with any NT was accurate (AUC>0.83 for all assays), but could be improved by changing the cutoff. Identifying samples with high NT could be achieved using an alternative cutoff.
ABSTRACT
Background. As the COVID-19 pandemic continues, growing attention has been placed on whether patients previously infected with SARS-CoV-2 have an increased risk of developing and/or exacerbating medical complications. Our study aimed to determine whether individuals with previous evidence of SARS-CoV-2 infection prior to their current emergency department (ED) visit were more likely to present with specific clinical sign/symptoms, laboratory markers, and/or clinical complications. Methods. A COVID-19 seroprevalence study was conducted at Johns Hopkins Hospital ED (JHH ED) from March 16 to May 31, 2020. Evidence of ever having SARSCoV-2 infection (PCR positive or IgG Ab positive) was found in 268 ED patients at this time (i.e. infected and/or previously infected). These patients were matched 1:2 to controls, by date, to other patients who attended the JHHED. Clinical signs/symptoms, laboratory markers, and/or clinical complications associated with ED visits and/ or hospitalizations at JHH within 6 months after their initial ED visit was ed through chart review for these 804 patients. Cox proportional hazards regression analyses were performed. Results. Among 804 ED patients analyzed, 50% were female, 56% Black race, and 15% Hispanic with a mean age of 47 years. 323 (40%) patients had at least 1 subsequent ED visit and additional 70 (9%) had been admitted to JHH. After controlling for race and ethnicity, patients with evidence of current or prior COVID-19 infection were more likely to require supplemental oxygen [hazards ratio (HR) =2.53;p=0.005] and have a cardiovascular complication [HR =2.13;p=0.008] during the subsequent ED visit than the non-infected patients. Conclusion. Our findings demonstrate that those previously infected with SARSCoV-2 have an increased frequency of cardiovascular complications and need for supplemental oxygen in ED visits in the months after their initial SARS-CoV-2 infection was detected. EDs could serve as a critical surveillance site for monitoring post-acute COVID-19 syndrome complications.
ABSTRACT
Background: The performance of serological antibody tests to SARS-CoV-2 infection varies widely and little is known about their performance in Africa. We assessed the performance of CoronaCHEK Lateral Flow Point of Care Tests on samples from Rakai, Uganda and Baltimore, Maryland, USA. Methods: Samples from subjects known to be SARS-CoV-2 PCR+ (Uganda: 50 samples from 50 individuals, and Baltimore: 266 samples from 38 individuals) and samples from pre-pandemic individuals collected prior to 2019 (Uganda: 1077 samples, Baltimore: 580 samples) were analyzed with the CoronaCHEK assay per manufacturers protocol. Sensitivity by duration of infection and specificity among pre-pandemic samples were assessed for the IgM and IgG bands separately and for any reactivity. Poisson regression models were used to calculate prevalence ratios (PR) for factors associated with a false-positive test among pre-pandemic samples. Results: In Baltimore samples, sensitivity for any reactivity increased with duration of infection with 39% (95% CI 30, 49) during 0-7 days since first positive PCR, 86% (95% CI 79, 92) for 8-14 days, and 100% (95% CI 89,100) after 15 days (See Figure). In Uganda, sensitivity was 100% (95% CI 61,100) during 0-7 days, 75% (95%CI 53, 89) for 8-14 days, and 87% (95%CI 55, 97) after 14 days since first positive PCR. Specificity results among pre-pandemic samples from Uganda was 96.5% (95% CI 97.5, 95.2), significantly lower than the 99.3% (95% CI 98.2, 99.8) observed in samples from Baltimore (p<0.01). In Ugandan samples, individuals with a false positive result were more likely to have had a fever more than a month prior to sample acquisition (PR 2.9, 95% CI 1.1, 7.0). Conclusion: Sensitivity of the CoronaCHEK appeared to be significantly higher in Ugandan samples from individuals within their first week of infection compared to their Baltimorean counterparts. By the second week of infection the sensitivity appeared the same between geographic areas. The specificity was significantly lower in Ugandan samples than those from Baltimore. False positive results from pre-pandemic Uganda appear to be correlated with the convalescent disease state, potentially indicative of a highly cross-reactive immune response in these individuals from East Africa.