The influence of time on the sensitivity of SARS-CoV-2 serological testing

Abstract

Sensitive serological testing is essential to estimate the proportion of the population exposed or infected with SARS-CoV-2, to guide booster vaccination and to select patients for treatment with anti-SARS-CoV-2 antibodies. The performance of serological tests is usually evaluated at 14-21 days post infection. This approach fails to take account of the important effect of time on test performance after infection or exposure has occurred. We performed parallel serological testing using 4 widely used assays (a multiplexed SARS-CoV-2 Nucleoprotein (N), Spike (S) and Receptor Binding Domain assay from Meso Scale Discovery (MSD), the Roche Elecsys-Nucleoprotein (Roche-N) and Spike (Roche-S) assays and the Abbott Nucleoprotein assay (Abbott-N) on serial positive monthly samples collected as part of the Co-STARs study ( www.clinicaltrials.gov , NCT04380896) up to 200 days following infection. Our findings demonstrate the considerable effect of time since symptom onset on the diagnostic sensitivity of different assays. Using a time-to-event analysis, we demonstrated that 50% of the Abbott nucleoprotein assays will give a negative result after 175 days (median survival time 95% CI 168-185 days), compared to the better performance over time of the Roche Elecsys nucleoprotein assay (93% survival probability at 200 days, 95% CI 88-97%). Assays targeting the spike protein showed a lower decline over the follow-up period, both for the MSD spike assay (97% survival probability at 200 days, 95% CI 95-99%) and the Roche Elecsys spike assay (95% survival probability at 200 days, 95% CI 93-97%). The best performing quantitative Roche Elecsys Spike assay showed no evidence of waning Spike antibody titers over the 200-day time course of the study. We have shown that compared to other assays evaluated, the Abbott-N assay fails to detect SARS-CoV-2 antibodies as time passes since infection. In contrast the Roche Elecsys Spike Assay and the MSD assay maintained a high sensitivity for the 200-day duration of the study. These limitations of the Abbott assay should be considered when quantifying the immune correlates of protection or the need for SARS-CoV-2 antibody therapy. The high levels of maintained detectable neutralizing spike antibody titers identified by the quantitative Roche Elecsys assay is encouraging and provides further evidence in support of long-lasting SARS-CoV-2 protection following natural infection.

Figure 1

Log transformed serial serological antibody titer data plotted by time from symptom onset. Antibody dynamics are dependent on the assay used with the sensitive Roche-S and MSD-S assay demonstrating maintenance of the spike protein antibody while the nucleoprotein antibody is shown to wane with the MSD and Abbott-N assays but to a lesser extent with the Roche-N assay.

Figure 2

Comparison of seropositivity and antibody dynamics between serological tests. The Roche-S assay target the spike antibody, the Abbott-N and the Roche-N assays target the N-antibody while the MSD assay targets the N-, the S- and the antibody to the Receptor Binding Domain (RBD) of the spike protein in parallel. (a) Kaplan–Meier curve and numbers at risk (the number of participants under follow up with serological tests available for analysis at that time point) for different serological tests. Y-axis represents the probability of remaining seropositive, while the X-axis shows days after symptom onset with numbers of participants under follow up shown in the table below. (b) Inferred posterior density distributions of the decay rate in a generalized linear mixed model.

Figure 3

Comparison of antibody titers between the Abbott-N assay and the MSD-N assay. (a) The quantitative results for the MSD-N assay were compared to those of the Abbott-N test for each sample taken. Colours divide the samples depending on whether it was positive (green) or negative (red) for the MSD-N assay. Dotted red lines represent the seropositivity threshold for the Abbott-N assay (horizontal) and the MSD-N test (vertical). (b) ROC curve for the Abbott-N assay using the MSD-N test as gold standard. The x ~ y line represents the profile of a random classifier. Blue shaded area shows the 95% CI.