Prevalence of SARS-CoV-2 antibodies in healthy blood donors from the state of Tyrol, Austria, in summer 2020

Between 8 June and 4 September 2020, we recruited blood donors at local blood donation events to take part in this epidemiologic study. To be eligible for participation, donors had to (i) meet the general requirements for a blood donation; (ii) be living in Tyrol; (iii) be in a healthy state (for instance, free of malignant diseases, autoimmune diseases or infectious diseases); and (iv) be asymptomatic for at least 28 days in case they had had a PCR-confirmed or suspected SARS-CoV‑2 infection. Out of 7244 potentially eligible individuals, a total of 5345 individuals took part in the study, corresponding to a participation rate of 73.8%. Blood donors of a broad age range (18–71 years) and of both sexes were included. The present study was approved by the local ethics committee of the Medical University Innsbruck (1134/2020) and participants provided written informed consent.

All participants of the study were asked to complete a 2-page questionnaire at the time of blood donation. The questionnaire included information on age, sex, place of residence, blood group, and intake of medication during the preceding 4 weeks. We also gathered information on whether participants had travelled abroad in the 6 months preceding the blood donation, or whether they had visited any COVID-19 hotspot areas in Austria or other Austrian federal states since 1 December 2019. Furthermore, we asked participants to report whether (and if yes, when) they thought they had gone through a SARS-CoV‑2 infection, whether infection was confirmed with PCR, diagnosed by a medical doctor, or suspected by the affected individual. The term “suspected infection” is used if people thought to have gone through a SARS-CoV‑2 infection because of the symptoms they had or if they were in contact with a confirmed SARS-CoV‑2 case.

We also conducted a telephone survey in a subset of study participants in order to compare the spectrum and duration of symptoms. Symptoms queried included cough, sore throat, limb pain, shortness of breath, dyspnea, headache, vomiting/nausea, diarrhea, anosmia and/or ageusia. Other questions recorded were need for further treatment, such as hospitalization, ICU treatment or if a medical doctor was consulted due to severity of symptoms. Finally, the outcome of prior antibody and/or PCR testing was addressed.

Serum samples of blood donors were screened for SARS-CoV‑2 IgG antibodies with the Abbott Alinity i instrument using the Abbott SARS-CoV‑2 IgG assay (Abbott Ireland, Sligo, Ireland), a high-throughput chemiluminescent microparticle immunoassay (CMIA) used for qualitative detection of IgG antibodies against SARS-CoV‑2 nucleocapsid protein, according to the manufacturer’s instructions. The performance (specificity/sensitivity) of this assay was validated previously [9, 10]. According to the manufacturer’s information, a sensitivity of 100% (95% confidence interval, CI, 95.89–100.0%) in samples analyzed 14 days after symptom onset and a specificity of 99.63% (95% CI 99.05–99.90) [11] could be expected. When blood donors were tested positive in this first assay, the WANTAI SARS-CoV‑2 Ab enzyme-linked immunosorbent assay (ELISA, Beijing Wantai Biological Pharmacy Enterprise, Beijing, China), detecting total SARS-CoV‑2 antibodies (IgG and IgM) directed against the SARS-CoV‑2 spike protein, was performed according to manufacturer’s instructions. When both tests were positive, samples were additionally analyzed using the WANTAI SARS-CoV‑2 IgM ELISA (Beijing Wantai Biological Pharmacy Enterprise) according to manufacturer’s instructions and an in-house neutralization assay. The neutralization test was based on a replication defective vesicular stomatitis virus (VSV) vector, which is pseudotyped with a C-terminally truncated spike protein of SARS-CoV‑2 (VSV‑S [12]) and encodes green flourescent protein (GFP) as marker gene. Briefly, vectors were pre-incubated with fourfold dilutions of heat-inactivated donor samples for one hour and subsequently HEK293T cells stably overexpressing angiotensin I converting enzyme 2 were infected with these mixtures. After 16 h, infected cells, i.e. GFP positive cells, were counted using an ImmunoSpot S6 Ultra‑V reader and FluoroSpot software (CTL Europe, Bonn, Germany). The antibody dilution, which resulted in > 50% reduction of the number of GFP positive cells as compared to the virus only control was defined as neutralizing titer. Neutralization titers ≤ 1:4 were regarded as negative.

Data are given as absolute frequencies (n) and percentages (%). For comparisons of seropositivity between different groups, odds ratios (OR) and 95% confidence intervals (CI) were calculated using binary logistic regressions. All statistical analyses were performed using SPSS 26 (IBM Corp. Released 2019. IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY, USA) or Stata 16 (StataCorp. 2019, Stata Statistical Software: Release 16. College Station, TX, USA). P values < 0.05 were considered statistically significant.

Of the 5345 individuals screened, 165 were seropositive for SARS-CoV‑2 IgG antibodies (Fig. 1). This corresponds to a seroprevalence of 3.1% (95% CI 2.6–3.6%), which is 5.1-fold higher (95% CI 4.5–6.0%) than the case number identified by the health authorities in the state-wide testing program (0.6%; 4536 out of 757,634) [8].

We then conducted detailed additional laboratory analysis on the 165 positive samples (Fig. 1). First, we retested the positive samples using the WANTAI SARS-CoV-2-Ab ELISA, which detects both IgM and IgG antibodies directed against the spike protein. The majority of samples were again positive (134 out of 165; 81.2%), whereas one sample was borderline (0.6%) and 28 were negative (17.0%). Of the previously positive samples, two (1.2%) were not retested with the second assay due to technical problems. If only double-positive individuals (spike and nucleocapsid antibodies) were considered as positive, overall seroprevalence would be 2.5% (95% CI 2.1–3.0%).

Second, we retested the 165 positive samples using the WANTAI SARS-CoV-2-IgM ELISA in order to resolve the percentage of blood donors harboring IgM antibodies directed against the spike protein of SARS-CoV‑2. This test showed that 65 samples were IgM antibody positive (39.4%), 10 were borderline (6.1%), and 82 were IgM antibody negative (49.7%). Due to technical problems 8 (4.8%) of the double-positive samples could not be analyzed for IgM antibodies.

Finally, in the 134 samples that were positive with the Abbott SARS-CoV‑2 IgG CMIA and the WANTAI SARS-CoV-2-Ab ELISA, we performed an in-house neutralization assay to investigate if the antibodies are capable of neutralizing SARS-CoV‑2. All the samples we tested in the neutralization assay showed a titer of at least 1:16 or higher. In total, 48 (35.8%) showed a neutralization titer of 1:16, 51 (38.1%) of 1:64, 28 (20.9%) of 1:256, 3 (2.2%) of ≥ 1:1024, and 4 were not tested.

Fig. 2 shows SARS-CoV‑2 IgG seroprevalences and odds ratios by age, sex and blood type. Among 2497 female and 2848 male blood donors, 76 (3.0%) and 89 (3.1%) were tested positive for SARS-CoV‑2 IgG antibodies, respectively. Seropositivity was not significantly different (OR = 1.03, 95% CI 0.75–1.40, P = 0.864) between female and male individuals. When we analyzed different age groups, the odds of being IgG seropositive was 1.62-fold higher in participants aged < 25 years than in the reference group of participants aged 45–54 years (95% CI 1.01–2.59; P = 0.043; 4.7% seropositive vs. 2.9% seropositive), but we observed no significant differences in seropositivity in the other age groups of 25–34 years, 35–44 years, 55–64 years and ≥ 65 years, compared to reference group. Seroprevalence was also similar when we grouped participants according to their blood type (all P > 0.05).

We analyzed SARS-CoV‑2 IgG seroprevalence among blood donors according to their places of residence grouped by the nine districts of Tyrol (Fig. 3). While the odds of seropositivity did not differ for most districts, we observed a highly elevated seroprevalence in the district of Landeck (16.6%). When compared to the district of Innsbruck (seroprevalence 2.0%), the odds ratio for seropositivity was 9.56 (95% CI 4.25–21.49, P < 0.001). In a secondary analysis restricted to the hotspot region Ischgl (which lies in the district of Landeck), 30 out of 91 blood donors were seropositive, corresponding to a seroprevalence of 33.0%.

We compared seroprevalence between participants who reported to have travelled and participants who reported to have not travelled, distinguishing between three types of travel destinations (all shown in Fig. 4). First, we assessed seroprevalence by self-reported travel to certain hotspot regions since 1 December 2019, excluding participants that permanently lived in these hotspot regions. In this analysis, we found a higher seroprevalence among the 244 participants reporting to have visited Ischgl (7.0%) than the 5101 participants who did not (2.9%), corresponding to an odds ratio for seropositivity of 2.51 (95% CI 1.49–4.21, P = 0.001). The odds ratios for travel to any of the other hotspot regions were not significant, although statistical power was limited for some due to small group sizes (e.g. Altenmarkt, Gasteinertal).

Second, we analyzed whether seroprevalence differed by self-reported travel to other Austrian federal states since 1 December 2019. Seroprevalence was 3.8% among the 1429 participants who travelled to other Austrian federal states and 2.8% among the 3916 participants who did not, corresponding to an odds ratio for seropositivity of 1.39 (95% CI 1.00–1.93, P = 0.052). Results for the individual federal states of Austria are shown in Fig. 4. While odds to be seropositive were elevated among participants with a recent travel to Carinthia (OR: 2.07, 95% CI 1.22–3.51, P < 0.007), they did not differ significantly by travel history to other federal states.

Third, we evaluated whether participants travelled abroad in the 6 months preceding the blood donation. Seroprevalence was 6.4% among the 467 participants reported to have travelled abroad and 2.8% among the 4878 participants who did not, corresponding to an odds ratio for seropositivity of 2.41 (95% CI 1.61–3.63, P < 0.001). The most common travel destination was Germany, associated with an odds ratio of 3.55 (95% CI 1.92–6.57, P < 0.001) of being seropositive (Fig. 4).

To evaluate if the intake of certain medications was associated with higher or lower prevalence, we assessed medication intake categorized by predefined different drug classes. When we compared seroprevalence according to intake of medication, we observed no significant differences by any of the drug classes angiotensin converting enzyme inhibitors/angiotensin receptor blockers, other antihypertensive drugs, analgesics, low-dose acetylsalicylic acid, lipid lowering agents, thyroid hormones, anti-allergic drugs and hormone preparations (i.e. hormonal contraception, postmenopausal hormone replacement therapy, all P > 0.05) (Fig. 4).

We conducted a telephone survey to assess self-reported symptoms among 123 participants who were seropositive and 122 participants who were seronegative and had suspected having had an infection or had a laboratory confirmed SARS-CoV‑2 infection in the past. The telephone survey covered the symptoms fever (> 38 °C), cough, sore throat, limb pain, shortness of breath, dyspnea, headache, vomiting/nausea, diarrhea, anosmia and ageusia. Of them, anosmia (OR = 2.49, 95% CI 1.32–4.68, P = 0.005) and ageusia (OR = 2.76, 95% CI 1.54–4.92, P = 0.001) were linked to higher odds of being seropositive, whereas cough (OR = 0.39, 95% CI 0.23–0.67, P = 0.001) and limb pain (OR = 0.51, 95% CI 0.30–0.86, P = 0.011) were linked to lower odds of being seropositive (Fig. 6). Out of the 123 seropositive participants, 30 reported none of the aforementioned symptoms (24.4%).