key: cord-0769351-6jc95tcr
authors: El-Khoury, Joe M; Schulz, Wade L; Durant, Thomas J S
title: Longitudinal Assessment of SARS-CoV-2 Anti-Nucleocapsid and anti-Spike-1-RBD Antibody Testing Following PCR-Detected SARS-CoV-2 Infection
date: 2021-04-05
journal: J Appl Lab Med
DOI: 10.1093/jalm/jfab030
sha: d8ed585fba1219f11cab95997cae6f77cc6d93f3
doc_id: 769351
cord_uid: 6jc95tcr

BACKGROUND: SARS-CoV-2 serologic assays are becoming increasingly available and may serve as a diagnostic aid in a multitude of settings relating to past infection status. However, there is limited literature detailing the longitudinal performance of EUA-cleared serologic assays in US populations, particularly in cohorts with a remote history of PCR-confirmed SARS-CoV-2 infection (e.g., > 2 months). METHODS: We evaluated the diagnostic sensitivities and specificities of the Elecsys® Anti-SARS-CoV-2 (anti-N) and Elecsys® Anti-SARS-CoV-2 S (anti-S1-RBD) assays, using 174 residual clinical samples up to 267 days post-PCR diagnosis of SARS-CoV-2 infection (n = 154) and a subset of samples obtained prior to the COVID-19 pandemic as negative controls (n = 20). RESULTS: The calculated diagnostic sensitivities for the anti-N and anti-S1-RBD assays were 89% and 93%, respectively. Of the 154 samples in the SARS-CoV-2-positive cohort, there were 6 discrepant results between the anti-N and anti-S1-RBD assays, 5 of which were specimens collected ≥ 200 days post-PCR positivity and only had detectable levels of anti-S1-RBD antibodies. When only considering specimens collected ≥ 100 days post-PCR positivity (n = 41), the sensitivities for the anti-N and anti-S1-RBD assays were 85% and 98%, respectively. CONCLUSIONS: The anti-S1-RBD assay demonstrated superior sensitivity at time points more remote to the PCR detection date, with 6 more specimens from the SARS-CoV-2-positive cohort detected, 5 of which were collected more than 200 days post-PCR positivity. While analytical differences and reagent lot-to-lot variability are possible, this may indicate that, in some instances, anti-S1-RBD antibodies may persist longer in vivo and may be a better target for detecting remote SARS-CoV-2 infection.

In this focused report, we characterize the longitudinal performance of two clinical serologic assays, detecting anti-N and anti-S1-RBD antibodies, using 154 residual clinical samples up to 267 days post-PCR diagnosis of SARS-CoV-2 infection and a subset of samples obtained prior to the COVID-19 pandemic as negative controls. We found that the assays evaluated in this report are highly sensitive and specific and remain positive in most specimens up to 267 days post-PCR detection of SARS-CoV-2 infection. Lastly, the Elecsys® Anti-SARS-CoV-2 S (anti-S1-RBD) assay was found to be slightly more sensitive when analyzing specimens drawn ≥ 100 days post-PCR confirmation.

In the ongoing COVID-19 pandemic, molecular-based diagnostics remain the gold standard for detecting acute infection (1) . Serologic assays are becoming increasingly available and may serve as a diagnostic aid in a multitude of settings relating to past infection status. The clinical utility of these tests remains part of an ongoing debate, but proposed indications include seroprevalence studies, the detection of asymptomatic infections, vaccine responsiveness, and cases where clinical suspicion of past or present SARS-CoV-2 infection is high, but PCR is negative (2, 3) . Currently, there is limited literature detailing the performance of emergency use authorization (EUA)-cleared serologic assays in US populations, particularly in cohorts with a remote history of PCR-confirmed SARS-CoV-2 infection (e.g., ≥ 2 months) (4-7). As a result, interpretation of these assays can be challenging when there is limited data to advise clinicians as to how long antibody tests can be expected to be positive in the general population.

Accordingly, there remains a significant need for additional investigation with respect to this, and the longitudinal assessment of antibody assay performance metrics.

Among the currently available EUA-cleared assays, there are a multitude of method formats which measure different antibody isotypes and target a variety of SARS-CoV-2 epitopes (4). Recently, two assays have been cleared through the EUA pathway which offer; (1) the qualitative measurement of high affinity total antibodies directed against the nucleocapsid (N) antigen (Elecsys® Anti-SARS-CoV-2) (Roche Diagnostics; Rotkreuz, Switzerland) and (2) the semi-quantitative measurement of high affinity total antibodies directed against receptor binding domain (RBD) portion of the spike (S) protein on the S1 subunit (Elecsys® Anti-SARS-CoV-2 S) (Roche Diagnostics; Rotkreuz, Switzerland). To our knowledge, and at the time of this writing, there are only two publications that investigate the persistence of anti-S1-RBD antibodies at time points ≥ 2-3 months post-PCR positivity (4, 5) . In this focused report, we present data from our internal clinical validation experiments which sought to characterize the longitudinal performance of two commercially available, EUA-cleared assays, targeting anti-S1-RBD and anti-N antibodies, up to 267 days post-PCR diagnosis of SARS-CoV-2 infection.

This study was reviewed and approved by the Yale University Internal Review Board as an exempt study using deidentified clinical excess specimens (IRB#2000028760). De-identified residual patient plasma samples were retrospectively identified for this project using previously described methods (8) (9) (10) .

Residual plasma samples were obtained from two cohorts: (1) LGT specimens were accessioned, immediately centrifuged following, and ordered tests were run off the primary specimen container. Following completion of testing, LGT containers were temporarily stored in a temperaturecontrolled output buffer for 6 hours and were subsequently transferred to a refrigerator (2-8 o C) for short term storage (5-7 days) until discard. Prior to discard, these LGT containers were identified (10), plasma was aliquoted, and refrigerated (2-8 o C).

SARS-CoV-2-negative samples were collected prior to the first positive SARS-CoV-2 PCR test at our institution (March 2020). Residual plasma samples from the SARS-CoV-2-negative cohort were originally collected for amino acid profile testing. These samples were collected in dark green-top (sodium heparin) containers (Becton Dickinson; Franklin Lakes, NJ). As part of routine clinical workflow, these specimens were transported to the laboratory on ice and upon receipt, immediately aliquoted and stored in a freezer (-20 o C). Samples were subsequently thawed for amino acid profile testing and then refrozen. Accordingly, the SARS-CoV-2-negative plasma samples included for this project were likely subjected to two freeze-thaw cycles.

Aliquoted plasma specimens were analyzed using the Elecsys® Anti-SARS-CoV-2 and the Elecsys® Anti-SARS-CoV-2 S, which measure anti-N and anti-S1-RBD total antibodies, respectively. Both assays use a double-antigen sandwich (DAGS) electrochemiluminescence format. The anti-S1-RBD assay is a semiquantitative assay which has an analytic measurement range (AMR) of 0.40-250 U/mL, and a clinical reportable range (CRR) up to 2500 U/mL using an on-board 1:10 dilution. A concentration ≥ 0.80 U/mL is considered 'reactive' whereas < 0.80 U/mL is considered 'non-reactive'. The anti-N assay is a qualitative assay wherein a cut-off index ≥ 1 is considered 'reactive'. All serologic testing was performed on a COBAS e801 (Roche Diagnostics; Risch-Rotkreuz, Switzerland) and in a manner that is consistent with the manufacturer's instructions and recommended cutoffs. Both assays were also validated with respect to method correlation, linearity (for anti-S1-RBD), reference range verification, freeze-thaw stability over two freeze-thaw cycles, inter-precision, and intra-precision (data not shown). Lastly, a Fisher's exact test was used to calculate confidence intervals for specificity and sensitivity.

A total of 174 residual plasma specimens were included in the final analysis, 20 of which were from the Both serologic assays were non-reactive for all specimens (n=20) from the SARS-CoV-2-negative cohort with a calculated diagnostic specificity of 100% (95% CI 84-100%). In the SARS-CoV-2-positive cohort, 17 specimens were non-reactive for anti-N antibodies, yielding a diagnostic sensitivity of 89% (95% CI 82-93%) ( Figure 1A) . The anti-S1-RBD assay was non-reactive for a total of 11 specimens in the SARS-CoV-2-positive cohort, yielding a diagnostic sensitivity of 93% (95% CI 88-96%) ( Figure 1B) 

While there are several publications detailing serologic kinetics and test performance during the initial 2-4 weeks following SARS-CoV-2 infection, there is little published data which describe assay performance metrics ≥ 2-3 months after infection (11, 12) . In this focused report, we describe the longitudinal performance characteristics of two automated serology assays up to 267 days post-PCR positivity. Throughout the post-PCR positivity specimen collection date range, the anti-S1-RBD assay demonstrated superior sensitivity, detecting 6 more specimens in the SARS-CoV-2-positive cohort relative to the anti-N assay, 5 of which were collected more than 200 days post-PCR positivity. When, only considering specimens collected ≥ 100 days post-PCR positivity (n=41), the calculated sensitivity for the anti-N and anti-S1-RBD assays was 85% (95% CI 71-93%) and 98% (95% CI 87-100%), respectively.

Both assays evaluated in this report use the same antibody detection format (DAGS) and share the same manufacturer. While analytical differences and reagent lot-to-lot variability are possible, our findings may indicate that, in some instances, anti-S1-RBD antibodies may persist and be detectable longer in vivo and may be a better target for identifying remote SARS-CoV-2 infection.

Previously published literature has shown that, in most patients, the initial serologic response following SARS-CoV-2 infection will occur within two weeks post-symptom onset (3, 12, 13) . Of note, the claimed sensitivities by the manufacturer for the anti-N and anti-S1-RBD assays for specimens collected ≥ 14-15 days post-PCR confirmation were 99.5 and 96.6%, respectively. During this timeframe postinfection, recent peer-reviewed literature described observed sensitivities for the Elecsys® Anti-SARS-CoV-2 (anti-N) and the Elecsys® Anti-SARS-CoV-2 S (anti-S1-RBD) assays to be around 90% (13, 14) . This is consistent with our findings, wherein our calculated sensitivity for anti-N and anti-S1-RBD between 2- Accordingly, these discrepancies can yield differences between observed and expected serologic testing results. An additional limitation is the small sample size in the SARS-CoV-2-negative cohort (n=20) which limits the generalizability of the specificity results. 

RBD) assays demonstrated good sensitivity throughout the observation period

S1-RBD) assay was found to be slightly more sensitive when analyzing specimens drawn ≥ 100 days post-PCR confirmation, although the differences observed were not statistically significant. These data are useful in guiding the clinical interpretation of serologic testing in patients who may have a remote history of SARS-CoV-2 infection

Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19

Serology for SARS-CoV-2: Apprehensions, opportunities, and the path forward

The Role of Antibody Testing for SARS-CoV-2: Is There One?

Characterization of a Pan-Immunoglobulin Assay Quantifying Antibodies Directed against the Receptor Binding Domain of the SARS-CoV-2 S1-Subunit of the Spike Protein: A Population-Based Study

Persisting Neutralizing Activity to SARS-CoV-2 over Months in Sera of COVID-19 Patients

Clinical Performance of the Elecsys Electrochemiluminescent Immunoassay for the Detection of SARS-CoV-2 Total Antibodies

Multi-Platform Comparison of SARS-CoV-2 Serology Assays for the Detection of COVID-19

Clinical Characteristics and Outcomes for 7,995 Patients with SARS-CoV-2

Bridging the Collaboration Gap: Real-time Identification of Clinical Specimens for Biomedical Research

Health care and precision medicine research: analysis of a scalable data science platform

Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19)

Antibody Responses to SARS-CoV-2 in Patients With Novel Coronavirus Disease

Head-to-Head Comparison of Two SARS-CoV-2 Serology Assays

Quantitative measurement of anti-SARS-CoV-2 antibodies: Analytical and clinical evaluation

Figure 2: Distribution of quantitative signal (y-axis, log scale) for anti-N and anti-S1-RBD antibody assays plotted as a function of days post-SARS-CoV-2 PCR positivity

Red dashed line represents the positivity threshold (≥ 1.0 COI) and the blue dashed line represents the upper limit of quantitation (250 COI)

Red dashed line represents the positivity threshold (≥ 0.8 U/mL) and the blue dashed line represents the upper limit of quantitation (2500 U/mL)