key: cord-0729274-xzpvupms authors: Erikstrup, Christian; Hother, Christoffer Egeberg; Pedersen, Ole Birger Vestager; Mølbak, Kåre; Skov, Robert Leo; Holm, Dorte Kinggaard; Sækmose, Susanne Gjørup; Nilsson, Anna Christine; Brooks, Patrick Terrence; Boldsen, Jens Kjærgaard; Mikkelsen, Christina; Gybel-Brask, Mikkel; Sørensen, Erik; Dinh, Khoa Manh; Mikkelsen, Susan; Møller, Bjarne Kuno; Haunstrup, Thure; Harritshøj, Lene; Jensen, Bitten Aagaard; Hjalgrim, Henrik; Lillevang, Søren Thue; Ullum, Henrik title: Estimation of SARS-CoV-2 infection fatality rate by real-time antibody screening of blood donors date: 2020-06-25 journal: Clin Infect Dis DOI: 10.1093/cid/ciaa849 sha: c355a3b2b1367658e2abd93547b3fb608ee31a05 doc_id: 729274 cord_uid: xzpvupms BACKGROUND: The pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has tremendous consequences for our societies. Knowledge of the seroprevalence of SARS-CoV-2 is needed to accurately monitor the spread of the epidemic and to calculate the infection fatality rate (IFR). These measures may help the authorities to make informed decisions and adjust the current societal interventions. The objective was to perform nationwide real-time seroprevalence surveying among blood donors as a tool to estimate previous SARS-CoV-2 infections and the population based IFR. METHODS: Danish blood donors aged 17–69 years giving blood April 6 to May 3 were tested for SARS-CoV-2 immunoglobulin M and G antibodies using a commercial lateral flow test. Antibody status was compared between geographical areas and an estimate of the IFR was calculated. The seroprevalence was adjusted for assay sensitivity and specificity taking the uncertainties of the test validation into account when reporting the 95% confidence intervals (CI). RESULTS: The first 20,640 blood donors were tested and a combined adjusted seroprevalence of 1.9% (CI: 0.8-2.3) was calculated. The seroprevalence differed across areas. Using available data on fatalities and population numbers a combined IFR in patients younger than 70 is estimated at 89 per 100,000 (CI: 72-211) infections. CONCLUSIONS: The IFR was estimated to be slightly lower than previously reported from other countries not using seroprevalence data. The IFR is likely several fold lower than the current estimate. We have initiated real-time nationwide anti-SARS-CoV-2 seroprevalence surveying of blood donations as a tool in monitoring the epidemic. Humanity is suffering from a pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The local severity of the epidemic and experiences from other countries are used by the health authorities to calibrate societal interventions. These interventions, e.g. the closing of schools, public institutions, prohibition of group gatherings, and even curfew, have tremendous consequences. The authorities rely on accurate real-time data to make informed decisions. Thus, numbers of patients tested positive for SARS-CoV-2, admitted to hospital, needing respiratory assistance or deceased from coronavirus disease 2019 (COVID-19) are updated on a daily basis. In contrast, little information exists on the percentage of the population with previous mild or asymptomatic . The proportion of the population who have overcome the infection can probably be approximated by testing for antibodies against SARS-CoV-2. Antibodies may confer immunity to repeat infection and a high proportion of immune individuals can attenuate the epidemic. Measures of anti-SARS-CoV-2 seroprevalence can also be used to estimate the clinical impact of COVID-19. Statistics on COVID-19 morbidity and mortality vary greatly due to varying testing strategies and e.g. the capacity of the health care system to treat infected patients [1] . Countries that diagnose mild infections will report lower morbidity and mortality compared to those with a less comprehensive testing strategy. An accurate measure of seroprevalence can be used to estimate the accumulated number of SARS-CoV-2 infections and thus the infection fatality rate (IFR) in the underlying population. Blood donors comprise approximately 4.7% of the Danish population in the same age group [2]. Healthy volunteers donate blood in all areas of the country ensuring wide geographical coverage. We have initiated a prospective screening of all blood donations for SARS-CoV-2 antibodies to establish a real-time nationwide overview of antibody status. The objective of this study is to perform a seroprevalence survey among blood donors as a tool in the monitoring of the SARS-CoV-2 epidemic. A c c e p t e d M a n u s c r i p t 6 In Denmark, approximately 270,000 blood donations are given annually. All Danish blood donation facilities participated in this survey. From April 6 to May 3, 2020 a total of 20,640 blood donations were given by 17-69-year-old donors. Blood donors are healthy and must comply with strict eligibility criteria [3] . Currently, donors must self-defer for two weeks if they develop fever with upper respiratory symptoms. The first patient with COVID-19 in Denmark was diagnosed on February 26, 2020. Subsequently, diagnostic testing for SARS-CoV-2 viral RNA was primarily performed in symptomatic individuals returning from high risk areas. On March 3, the government recommended home-quarantine for 14 days in case of exposure to COVID-19 including traveling to high-risk areas and on March 6, events with more than 1,000 individuals were recommended to be postponed or cancelled. On March 11, the government established a partial lockdown of the country: for public employees only persons with critical functions were allowed to work, schools and child care facilities were closed except when parents served in critical functions, workers in the private sector were recommended to work from home, and gatherings of more than 100 were prohibited. Validation and testing were performed by experienced staff in five regional blood establishments. We retrieved data on population numbers as of January 1 st 2020 [5] and the number of infected and deceased due to COVID-19 using daily updated data [6] . Statistical analysis was performed in RStudio 1.2 and R 3.6.0. Results were reported as percentages with 95% confidence intervals (CI). The EpiR package was used to adjust seroprevalence for sensitivity and specificity. We used the Rogan Gladen estimate to calculate the true prevalence. CI were derived by 10^8sample percentile bootstrapping independently sampling sensitivity, specificity and apparent prevalence using posterior binomial distributions. A c c e p t e d M a n u s c r i p t 8 Ethics SARS-CoV-2 antibody testing was performed as a routine screening of all blood donations. Only consenting donors were tested and informed about their result. Anonymized data was used in this study. The Regional Scientific Ethical Committees for the Zealand Region of Denmark approved the investigation as a register project (20-000013). We included blood donors aged 17 to 69 years and a total of 20,640 blood donors were informed and all consented to testing; see Table 1 for characteristics. The distribution between seropositivity for IgM and IgG appears in Table 2 . The estimated number of infected individuals was calculated per area in the relevant age group ( Table 3) . The overall unadjusted seroprevalence was 2.0% (CI: 1.8-2.2). After adjusting for assay sensitivity and specificity including their CI, the overall seroprevalence was 1.9% (CI: 0.8-2.3). The seroprevalence in the Capital Region was higher than in the other four regions combined (3.2% vs 1.2%, difference: 2.0 percentage points, CI: 1.4-2.6). There was an insignificant increase in seroprevalence from the first two weeks to the last two weeks (1.7% vs 2.0%, difference: 0.28 percentage points, CI: -0.15-0.78). In this survey of SARS-CoV-2 antibodies in Danish blood donors we found a seroprevalence of 1.9 (CI: 0.8-2.3) adjusted for the assay performance and a low IFR of 89/100,000 (CI: 72-211). This IFR of 0.089% is slightly lower than a recently published COVID-19 IFR estimate of 0.145% (CI: 0.088-0.317, individuals below 60 years) not including seroprevalence data [7] . The ratio between estimated antibody-positive individuals and confirmed COVID-19 cases is expected given the targeted early Danish SARS-CoV-2 testing strategy. The lack of large seroprevalence surveys prevents a comparison with other areas/countries. The low IFR is encouraging, but several caveats exist. Although blood donors represent a very broad population base, they are selected healthy and self-defer for two weeks after signs of COVID-19. Conversely, blood donor prevalence increases with income [8] and we speculate that this leads to higher risk of exposure through travel and social activity. We may therefore either under or overestimate the true population immunity. We validated the antibody assay primarily in individuals diagnosed with clinical COVID-19. If silent and mild infections lead to weaker antibody responses, we will underestimate the population immunity. Conversely, we found that 42.7% of donors testing positive were IgM-only reactive. It is possible that some of these individuals had asymptomatic infection and we cannot rule out that some were infectious while reporting for donation. Also, screening only for antibodies may underestimate the prevalence of infections, if cellular cytotoxicity is able to eradicate virally infected cells, as for SARS-CoV, before eliciting a humoral response [9] . Finally, this study only addresses the IFR in 17-69-year-old individuals. The IFR in other population strata, e.g. among individuals above 80 or with comorbidity is higher [7, 10] . A c c e p t e d M a n u s c r i p t 10 Currently, the governments in most countries are trying to balance the economic consequences of a societal lockdown against the risk of an uncontrolled epidemic. Our results underpin that social distancing in a healthy population predominately acts as a means to protect vulnerable individuals. It would be challenging to perform an unbiased seroprevalence survey in the background population. As blood donation facilities are located nationwide and operate continuously the screening is suited to monitor regional differences and temporal changes. With greater knowledge of the seroprevalence in other population strata the continued monitoring may also be used to effectively model the activity of the SARS-CoV-2 epidemic. We undertook a validation and found a less than perfect sensitivity of 82.6% (75.7-88.2) when previous PCR-confirmed COVID-19 patients were tested. However, it is known that not all infected individuals produce antibodies. The specificity was acceptable at 99.5% (98.7-99.9) but leads to a low positive predictive value in low-prevalence areas. We used a conservative method to estimate the confidence interval and thus took not only the sample variation but also the uncertainty in the sensitivity and specificity into account. This is necessary because we, unlike most diagnostic and screening tests, do not have a Gold Standard to confirm positive or negative results. The confidence interval for the regions with lowest antibody prevalence thus reached a lower limit seroprevalence of 0%. We chose to use the current lateral flow test because of early availability enabling us to produce the first SARS-CoV-2 antibody seroprevalence estimate in Denmark. While the assay performed acceptably, we recommend to use a well-validated lab-based assay, which are now available, for subsequent seroprevalence studies. A c c e p t e d M a n u s c r i p t The estimates for the IFR should allow for the lag time from infection to death. Based on current literature time from infection to death in non-survivors is 23-30 days [11, 12] . Similarly, the lag time from infection to the detection of antibodies may be 16 days [11, 13] . Donor self-deferral due to respiratory symptoms will add to the lag time for the detection of antibodies. We used the last available total of deceased citizens due to COVID-19 on the last date of the study (May 3, 2020). Using earlier values would result in a lower IFR estimate while waiting for later death tolls would result in a higher IFR. The death toll among all citizens below 70 years was used even though only 20 of 65 deaths appeared among individuals with no comorbidity. This was chosen because the denominator included all citizens in the age strata, thus, also individuals with comorbidity. The IFR including only individuals with no comorbidity is thus likely several fold lower than the current estimate. Rapid tests are read by individuals and inter-observer variation often exist. Furthermore, there is uncertainty regarding cross reactivity of SARS-CoV-2 and other coronavirus antibodies. The results included in this article will be updated and freely accessible at http://www.bloddonor.dk/antisarscov2. Our results indicate that the IFR among individuals aged 17 to 69 years is 89/100,000 (CI: 72-211). This may have implications for risk mitigation. The IFR in older population strata may be considerably higher. Nationwide continuous seroprevalence surveying of blood donations may be a tool in monitoring the SARS- A c c e p t e d M a n u s c r i p t 12 M a n u s c r i p t 14 Tables Table 1 Female Male Age and sex stratified seroprevalence of anti-SARS-CoV-2. Likelihood of survival of coronavirus disease 2019 Blood donation and blood donor mortality after adjustment for a healthy donor effect Spring 2020 -transcripts in Danish Statistics Denmark -population data COVID-19 i Danmark Epidemiologisk overvågningsrapport 2020-04-13 Estimates of the severity of coronavirus disease 2019: a modelbased analysis Socio-demographic characteristics of Danish blood donors T cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice Severe Outcomes Among Patients with Coronavirus Disease 2019 (COVID-19) -United States The Incubation Period of Coronavirus Disease From Publicly Reported Confirmed Cases: Estimation and Application Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease Interpreting Diagnostic Tests for SARS-CoV-2 We thank laboratory technicians from the Departments of Clinical Immunology at Aarhus University Hospital, Copenhagen University Hospital, Odense University Hospital, Zealand University Hospital, and Aalborg University Hospital for their excellent work during the validation of the assay and testing of the samples. The Livzon tests were donated by BESTSELLER foundation. BESTSELLER foundation had no influence on the study. The authors report no conflict of interest. A c c e p t e d M a n u s c r i p t 13 M a n u s c r i p t 15 A c c e p t e d M a n u s c r i p t 16 [14] .