key: cord-0683194-5ji8em53
authors: Hønge, Bo Langhoff; Hindhede, Lotte; Kaspersen, Kathrine Agergård; Harritshøj, Lene Holm; Mikkelsen, Susan; Holm, Dorte Kinggaard; Nilsson, Anna Christine; Sækmose, Susanne Gjørup; Sørensen, Erik; Aagaard, Bitten; Hjalgrim, Henrik; Jørgensen, Charlotte Sværke; Krause, Tyra Grove; Ullum, Henrik; Pedersen, Ole Birger Vestager; Ostrowski, Sisse Rye; Erikstrup, Christian
title: Long-term detection of SARS-CoV-2 antibodies after infection and risk of re-infection
date: 2022-01-22
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2022.01.041
sha: 93b5c5c65e088e04dca8a22c75540e6bcef4005e
doc_id: 683194
cord_uid: 5ji8em53

Objectives : To evaluate long-term sensitivity for detection of total antibodies against SARS-CoV-2 Methods : From week 41, 2020 through week 26, 2021 all Danish blood donations were tested for SARS-CoV-2 antibodies with the Wantai assay. Results were linked with PCR test results from the Danish Microbiological Database (MiBa). Results : During the study period, 105,646 non-vaccinated Danish blood donors were tested for SARS-CoV-2 antibodies, and 3,806 (3.6%) had a positive PCR test prior to the blood donation. Among the donors with a positive PCR test, 94.2% also had a positive antibody test subsequently. Time between positive PCR test and antibody test were up to 15 months and there was no evidence of decline in proportion with detectable antibodies over time. A negative serological result test was associated with higher incidence of re-infection (Incidence Rate Ratio = 0.102 (95% CI: 0.039–0.262)). Conclusions : Among health blood donors 94.2% developed SARS-CoV-2 antibodies after infection, and lack of detectable antibodies was associated with re-infection.

A crucial, and much debated, issue for the current vaccination strategy against SARS-CoV-2 is the duration of immunity and detectable antibodies. Reports from the initial epicenter of the pandemic in Wuhan, China, show that individuals with previous SARS-CoV-2 infection have declining antibody titers during the first year after infection and that male gender and younger age are associated with higher IgG antibody titers (Feng et al., 2021) . Other factors may influence immunity such as comorbidity, immunosuppression, and genetic predisposition including blood type (Barnkob et al., 2020; Bastard et al., 2021; Cordtz et al., 2020) . It has not been thoroughly investigated why some otherwise healthy individuals do not produce detectable levels of antibodies or become re-infected in spite of detectable antibody levels.

Serological assays are used to demonstrate previous infection or vaccination response against SARS-CoV-2 and may guide health authorities planning measures against COVID-19 spread (Jones et al., 2021) . Assays detecting total immunoglobulin, i.e., both IgM, IgA, and IgG, are useful to assure maximum sensitivity (SARS-CoV, n.d.) . Several assay evaluations have been performed to estimate short-term sensitivity and in a comparison with 15 other serological assays, Wantai SARS-CoV-2 Ab ELISA assay (Wantai, Beijing, China) performed best with a sensitivity of 96.7% and a specificity of 99.5% (Harritshøj et al., 2021) . However, only few studies have explored the association between total immunoglobulin against SARS-CoV-2 and protective immunity.

We assessed the persistence of total anti-SARS-CoV-2 antibodies after real-time polymerase chain reaction (PCR) confirmed SARS-CoV-2 infection, risk factors for undetectable antibodies, and rate of re-infection among seropositive and seronegative individuals.

During the SARS-CoV-2 epidemic, more than 200,000 blood donations from Danish blood donors have been screened for SARS-CoV-2 antibodies to assist the Danish health authorities in the surveillance of the pandemic. Donor data from all five regions in Denmark (Pedersen et al., 2012) were included in this study.

From week 41, 2020 through week 26, 2021 all Danish blood donations were tested for SARS-CoV-2 antibodies. The Wantai SARS-CoV-2 Ab ELISA assays were performed locally in each of the five regional blood centers. Wantai is a qualitative assay detecting antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein.

Information about SARS-CoV-2 infection was obtained from the Danish Microbiological Database (MiBa). Individuals were considered SARS-CoV-2 infected if they had a positive PCR test. Data were available from the earliest case in February 2020 until August 2021. Blood donors were given a 28-day deferral period after a positive SARS-CoV-2 test.

The sensitivity of the Wantai assay was assessed in three-month periods. Individuals stating having been vaccinated against SARS-CoV-2 were excluded from this analysis from time of vaccination.

For individuals with more than one donation in one time period, only the last donation was included in the analysis. Sensitivity with ninety-five % confidence intervals (CIs) were calculated by bootstrapping 10 6 times. Binomial regression analysis was used to test for time-dependent loss of sensitivity and risk factors for a negative Wantai test among PCR positives were assessed by binomial regression. Donors were considered re-infected if they had two positive PCR tests at least three months apart. Poisson regression was used to calculate rate of re-infection. Estimated number of false positive tests among the re-infected donors was calculated by the following equation: (1specificity) x total number of tests x incidence of primary infection. Statistical analyses were performed using R 4.0.0 (R Foundation for Statistical Computing, Vienna, Austria).

Consenting blood donors were tested for SARS-CoV-2 antibodies and informed about the result.

The study was approved by the Regional Scientific Ethical Committee in Region Zealand, Denmark (SJ-740). Also, the study was approved by the Danish Data Protection Agency (P-2019-99).

According to Danish law, approval from a national ethical committee is not necessary when using administrative register data without individual contact.

During the study period, 105,646 non-vaccinated Danish blood donors were tested for SARS-CoV-2 antibodies, and 3,806 (3.6%) had a positive PCR test prior to the blood donation (Table 1) .

Among the donors with a positive PCR test, 3,587 also had a positive antibody test subsequently (overall sensitivity 94.2%). We found no correlation between positive antibody tests and days since infection (risk ratio = 1.00 (95% CI 1.00-1.00), P = 0.10), and there was no difference in antibody detection during the first and the second wave of COVID-19 in Denmark (p<0.01). In univariable analysis, neither sex, age, comorbidity, nor blood type were associated with a positive test for total antibodies after SARS-CoV-2 infection ( Table 1) . Of the 1,218 donors with two or more antibody tests 9 donors seroconverted from negative to positive, and 6 seroconverted from positive to negative.

Among individuals with a positive PCR test prior to blood donation, 21 (0.6%) had a second positive PCR test at least three months after the initial PCR test as a sign of re-infection, and 18 donors also had an antibody test result in between the two PCR tests. Among these individuals, 11 (61.1%) had detectable antibodies and the remaining seven had no detectable SARS-CoV-2 antibodies (negative antibody test result). The seven individuals with a negative antibody test only donated once in the study period and were therefore only antibody tested once. Average time between infections was 171.9 days, and average time between last antibody test and second positive PCR test was 65.7 days. The seven individuals with negative antibody tests were compared with the 11 individuals with a positive antibody test according to the characteristics listed in Table 1 . There were no differences between groups (exact data not shown due to confidentiality of small group sizes). The rate of documented re-infection was 440/100,000 person-years for seropositives and 4,332/100,000 person-years for seronegatives; Incidence Rate Ratio (IRR) = 0.102 (95% CI: 0.039-0.262). In comparison the rate of primary infection was 4,379/100,000 person-years for seronegatives (95% CI: 4,211-4,554). With an estimated PCR test specificity of 99.98% (Hansen et al., 2021) and an incidence rate of 4,379/100,000 person-years for seronegatives, we estimate that 1 out of the 7 "re-infected" individuals tested false positive initially.

In this study, we found a consistent and persistent high sensitivity of a total SARS-CoV-2 antibody assay, Wantai, up to 15 months after infection. Re-infection occurred in 0.6% of our study participants and was associated with having a negative antibody test.

Main strengths of this study are the large sample size and nationwide design using national health registers and the screening for SARS-CoV-2 antibodies in a large cohort of Danish blood donors.

After donation, blood donors had access to their serological results which may have affected their behavior. Most likely, individuals' social behavior is affected by a positive antibody result and they may have felt better protected against risk of SARS-CoV-2 re-infection and have engaged more in social activities compared to individuals with a negative antibody result.

for one year (Feng et al., 2021) . The current total immunoglobulin assay was previously documented to be sensitive for detection of antibodies a couple of months after infection. Longterm antibody detection of the Wantai assay have, however, only been evaluated in minor selected populations (Bal et al., 2021) and sensitivities above 95% have been reported.

A recent Danish study including all Danish citizens found that 0.65% of the population experienced re-infection with SARS-CoV-2 between first and second wave (Hansen et al., 2021 )a re-infection rate very similar to ours (0.6%) considering a higher infection rate among the 17 to 70 year old and longer follow-up period. This indicates that our results are generalizable to the target population.

We did not identify risk factors of a negative antibody test. This may partly be due to a large homogeneity in a blood donor population with regards to lack of co-morbidity. Also, we did not have knowledge on the severity of symptoms in individuals with a positive PCR test. Among Danish health care workers, a negative antibody test after verified SARS-CoV-2 infection was associated with self-reported asymptomatic infection or mild infection, and with body mass index ≥30 (Johannesen et al., 2021) . In a study by Petersen LR et al 6.3% of previously infected individuals did not develop detectable antibodies (Petersen et al., 2021) . Here, lack of antibodies was associated with asymptomatic infections, immunosuppressive medications, race/ethnicity, and obesity. In our study, we did not have information on disease severity to perform this evaluation.

Antibody response may also depend on non-individual factors such as inoculation dosage and viral burden (Walker et al., 2021) . Unfortunately, we did not have information on CT-levels for the PCR tests to make such an evaluation. We found no difference in test sensitivity during first and second wave where different SARS-CoV-2 (non-delta) variants were dominant indicating that viral strain difference had little impact on development of detectable antibodies. We estimated the number of "false positive" in the group of re-infected donors, but this estimate contain some uncertainty.

Presence of antibodies does not necessarily equal protective immunity, and antibody titers were not known. More than 60% of the re-infections reported here occurred among individuals with detectable antibodies. On the other hand, the risk of re-infection was more than 9 times higher among individuals without antibodies when compared with individuals with antibodies. Individuals with previous SARS-CoV-2 infection, but with undetectable antibodies, have approximately the same risk of re-infection as individuals without previous exposure to SARS-CoV-2 (Hansen et al., 2021 

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The authors declare no conflicts of interest.

This work was supported by Central Denmark Region. The Wantai tests were acquired by The Danish Health Authority requisitioned through Statens Serum Institut. The funders had no role in performing this study.