Detection of Nucleocapsid Antibodies Associated with Primary SARS-CoV-2 Infection in Unvaccinated and Vaccinated Blood Donors.

Nucleocapsid antibody assays can be used to estimate SARS-CoV-2 infection prevalence in regions implementing spike-based COVID-19 vaccines. However, poor sensitivity of nucleocapsid antibody assays in detecting infection after vaccination has been reported. We derived a lower cutoff for identifying previous infections in a large blood donor cohort (N = 142,599) by using the Ortho VITROS Anti-SARS-CoV-2 Total-N Antibody assay, improving sensitivity while maintaining specificity >98%. We validated sensitivity in samples donated after self-reported swab-confirmed infections diagnoses. Sensitivity for first infections in unvaccinated donors was 98.1% (95% CI 98.0-98.2) and for infection after vaccination was 95.6% (95% CI 95.6-95.7) based on the standard cutoff. Regression analysis showed sensitivity was reduced in the Delta compared with Omicron period, in older donors, in asymptomatic infections, <30 days after infection, and for infection after vaccination. The standard Ortho N antibody threshold demonstrated good sensitivity, which was modestly improved with the revised cutoff.

Selecting COVID-19 convalescent plasma for neutralizing antibody potency using a high-capacity SARS-CoV-2 antibody assay.

BACKGROUND: Efficacy of COVID-19 convalescent plasma (CCP) is hypothesized to be associated with the concentration of neutralizing antibodies (nAb) to SARS-CoV-2. High capacity serologic assays detecting binding antibodies (bAb) have been developed; nAb assays are not adaptable to high-throughput testing. We sought to determine the effectiveness of using surrogate bAb signal-to-cutoff ratios (S/Co) in predicting nAb titers using a pseudovirus reporter viral particle neutralization (RVPN) assay. METHODS: CCP donor serum collected by three US blood collectors was tested with a bAb assay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and a nAb RVPN assay. Prediction effectiveness of various CoV2T S/Co criteria was evaluated for RVPN nAb NT50 titers using receiver operating characteristics. RESULTS: Seven hundred and fifty-three CCPs were tested with median CoV2T S/Co and NT50 of 71.2 of 527.5. Proportions of donors with NT50 over target nAb titers were 86% ≥1:80, 76% ≥1:160, and 62% ≥1:320. Increasing CoV2T S/Co criterion reduced the sensitivity to predict NT50 titers, while specificity to identify those below increased. As target NT50 titers increase, the CoV2T assay becomes less accurate as a predictor with a decline in positive predictive value and rise in negative predictive value. CONCLUSION: Selection of a clinically effective nAb titer will impact availability of CCP. Product release with CoV2T assay S/Co criterion must balance the risk of releasing products below target nAb titers with the cost of false negatives. A two-step testing scheme may be optimal, with nAb testing on CoV2T samples with S/Cos below criterion.

Comparison of Detection Limits of Fourth- and Fifth-Generation Combination HIV Antigen-Antibody, p24 Antigen, and Viral Load Assays on Diverse HIV Isolates.

Detection of acute HIV infection is critical for HIV public health and diagnostics. Clinical fourth-generation antigen (Ag)/antibody (Ab) combination (combo) and p24 Ag immunoassays have enhanced detection of acute infection compared to Ab-alone assays but require ongoing evaluation with currently circulating diverse subtypes. Genetically and geographically diverse HIV clinical isolates were used to assess clinical HIV diagnostic, blood screening, and next-generation assays. Three-hundred-member panels of 20 serially diluted well-characterized antibody-negative HIV isolates for which the researchers were blind to the results (blind panels) were distributed to manufacturers and end-user labs to assess the relative analytic sensitivity of currently approved and preapproved clinical HIV fourth-generation Ag/Ab combo or p24 Ag-alone immunoassays for the detection of diverse subtypes. The limits of detection (LODs) of virus were estimated for different subtypes relative to confirmed viral loads. Analysis of immunoassay sensitivity was benchmarked against confirmed viral load measurements on the blind panel. On the basis of the proportion of positive results on 300 observations, all Ag/Ab combo and standard sensitivity p24 Ag assays performed similarly and within half-log LODs, illustrating the similar breadth of reactivity and diagnostic utility. Ultrasensitive p24 Ag assays achieved dramatically increased sensitivities, while the rapid combo assays performed poorly. The similar performance of the different commercially available fourth-generation assays on diverse subtypes supports their use in broad geographic settings with locally circulating HIV clades and recombinant strains. Next-generation preclinical ultrasensitive p24 Ag assays achieved dramatically improved sensitivity, while rapid fourth-generation assays performed poorly for p24 Ag detection.

Development and Implementation of Dried Blood Spot-Based COVID-19 Serological Assays for Epidemiologic Studies.

Serological surveillance studies of infectious diseases provide population-level estimates of infection and antibody prevalence, generating crucial insight into population-level immunity, risk factors leading to infection, and effectiveness of public health measures. These studies traditionally rely on detection of pathogen-specific antibodies in samples derived from venipuncture, an expensive and logistically challenging aspect of serological surveillance. During the COVID-19 pandemic, guidelines implemented to prevent the spread of SARS-CoV-2 infection made collection of venous blood logistically difficult at a time when SARS-CoV-2 serosurveillance was urgently needed. Dried blood spots (DBS) have generated interest as an alternative to venous blood for SARS-CoV-2 serological applications due to their stability, low cost, and ease of collection; DBS samples can be self-generated via fingerprick by community members and mailed at ambient temperatures. Here, we detail the development of four DBS-based SARS-CoV-2 serological methods and demonstrate their implementation in a large serological survey of community members from 12 cities in the East Bay region of the San Francisco metropolitan area using at-home DBS collection. We find that DBS perform similarly to plasma/serum in enzyme-linked immunosorbent assays and commercial SARS-CoV-2 serological assays. In addition, we show that DBS samples can reliably detect antibody responses months postinfection and track antibody kinetics after vaccination. Implementation of DBS enabled collection of valuable serological data from our study population to investigate changes in seroprevalence over an 8-month period. Our work makes a strong argument for the implementation of DBS in serological studies, not just for SARS-CoV-2, but any situation where phlebotomy is inaccessible. IMPORTANCE Estimation of community-level antibody responses to SARS-CoV-2 from infection or vaccination is critical to inform public health responses. Traditional studies of antibodies rely on collection of blood via venipuncture, an invasive procedure not amenable to pandemic-related social-distancing measures. Dried blood spots (DBS) are an alternative to venipuncture, since they can be self-collected by study participants at home and do not require refrigeration for shipment or storage. However, DBS-based assays to measure antibody levels to SARS-CoV-2 have not been widely utilized. Here, we show that DBS are comparable to blood as a sampling method for antibody responses to SARS-CoV-2 infection and vaccination over time measured using four distinct serological assays. The DBS format enabled antibody surveillance in a longitudinal cohort where study participants self-collected samples, ensuring the participants' safety during an ongoing pandemic. Our work demonstrates that DBS are an excellent sampling method for measuring antibody responses whenever venipuncture is impractical.

Analysis of SARS-CoV-2 antibodies in COVID-19 convalescent blood using a coronavirus antigen microarray.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Blood using a Coronavirus Antigen Microarray.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Evaluation of a new Trypanosoma cruzi antibody assay for blood donor screening.

BACKGROUND: This multicenter prospective study was designed to evaluate the performance characteristics of a new commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Trypanosoma cruzi in blood donors, the ORTHO T. cruzi ELISA Test System (Ortho-Clinical Diagnostics). STUDY DESIGN AND METHODS: Assay specificity was evaluated among 40,665 serum and ethylenediaminetetraacetate (EDTA) plasma specimens from volunteer blood donors and 481 T. cruzi antibody-negative specimens from a high-risk population. Sensitivity was evaluated among 106 T. cruzi-infected subjects identified by parasite detection, among 93 radioimmunoprecipitation assay (RIPA)-positive specimens from high-risk subjects, and 662 specimens presumed positive for the presence of T. cruzi antibodies by serologic methods. Also assessed were the equivalence of serum and plasma as specimen sources, performance equivalence of automated and semiautomated processing methods, nonspecific reactivity in specimens from other disease states or clinical conditions, and assay precision. RESULTS: Assay specificity was 99.998 percent in volunteer blood donors and 99.4 percent among high-risk subjects. Sensitivity was 100 percent among specimens positive by parasite detection, or by serologic methods, and 98.9 percent among RIPA-positive specimens from high-risk subjects. No differences were demonstrated between serum and plasma or between semiautomated and automated processing methods. Cross-reactivity was observed with known positive leishmaniasis specimens. Total inter- and intraassay variability was less than 10 percent with both the automated and the semiautomated methods. CONCLUSION: The ORTHO T. cruzi ELISA Test System is an effective, qualitative assay for screening blood donors for immunoglobulin G antibodies to T. cruzi. The assay was licensed for donor screening by the FDA in December 2006.

Evaluation of a new enzyme-linked immunosorbent assay for detection of Chagas antibody in US blood donors.

BACKGROUND: Chagas disease, caused by the parasite Trypanosoma cruzi, represents a serious blood safety problem due to increasing immigration from Latin America. The Food and Drug Administration recently recommended implementation of Chagas antibody screening for US donors as soon as a suitable assay is licensed. An anonymized preclinical study of a prototype T. cruzi lysate-based enzyme-linked immunosorbent assay (ELISA) developed by Ortho-Clinical Diagnostics was conducted. STUDY DESIGN AND METHODS: Two populations of specimens were evaluated: 1) 10,192 sequential donations from blood donors residing in the El Paso, Texas, area and 2) 178 specimens from South America which were presumptively positive for antibodies to T. cruzi and purchased from commercial vendors. RESULTS: A total of 10,189 (99.97%) of the 10,192 screened donor specimens did not react, whereas 3 (0.03%) tested initially reactive. The 3 initially reactive specimens tested repeat reactive and were confirmed by radioimmunoprecipitation analysis (RIPA). Based on antibody profile analysis, 2 of the 3 Chagas-positive specimens were from the same donor. Observed specificity of the test was therefore 100 percent. Of the specimens from South America, 173 of 178 were reactive by the prototype ELISA. Of the 5 nonreactive specimens, all did not react by indirect fluorescence assay, but 4 were positive by RIPA. Therefore, calculated sensitivity of the ELISA was 97.7 percent (173/177). CONCLUSIONS: These studies indicate that the prototype ELISA has excellent sensitivity and specificity for detection of antibodies to T. cruzi in donors. Moreover, among donations from a geographically selected collection region of the United States, observed seroprevalence was 0.03 percent.