key: cord-1029344-6495zddb authors: Kraehling, V.; Kern, M.; Halwe, S.; Mueller, H.; Rohde, C.; Savini, M.; Schmidt, M.; Wilhelm, J.; Becker, S.; Ciesek, S.; Gottschalk, R. title: Epidemiological study to detect active SARS-CoV-2 infections and seropositive persons in a selected cohort of employees in the Frankfurt am Main metropolitan area date: 2020-05-25 journal: nan DOI: 10.1101/2020.05.20.20107730 sha: 0c9fc431f7e00483e365bc29526afeb8930a824f doc_id: 1029344 cord_uid: 6495zddb So far, 170,000 Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections have been confirmed in Germany, of which more than 5,000 have been detected in the Frankfurt am Main metropolitan region. When examining 1,000 nasopharyngeal swabs and serum samples from healthy volunteers from this region, one RT-PCR-positive and five antibody-positive persons were identified. The five positive serum samples were confirmed to be specific. Four of the five positive sera cross-neutralized SARS-CoV. More than 4 million people have been infected with SARS-CoV-2 globally with nearly 280,000 fatalities. It is important for the containment of the SARS-CoV-2 pandemic to be aware of the total number of infected people, which is probably underestimated due to the underreporting of mild and asymptomatic cases. In preliminary work we examined symptom-free travelers for SARS-CoV-2specific RNA to detect unnoticed COVID-19 cases. Among 122 passengers, who returned from Wuhan in February 2020, we identified two infected asymptomatic individuals (data not shown). This result suggests that the screening of asymptomatic individuals could be helpful in estimating the actual number of acute infections in the population. Several reports indicated that a combination of Realtime PCR (RT-PCR) and serological assays can be superior to identifying SARS-CoV-2 compared to RT-PCR alone [1] [2] [3] . The project aims to determine the current SARS-CoV-2 status of a selected cohort of 1,000 employees in the Frankfurt metropolitan area to contribute to an accurate knowledge of the status of the SARS-CoV-2 pandemic. The study was announced to the employees of Infraserv Höchst, a large industrial site operator in Frankfurt am Main at the beginning of April 2020 by the company's own Occupational Health Center. All employees were allowed to participate until a cohort of 1,000 subjects was reached. There were no exclusion criteria. Nasopharyngeal swab and blood samples (serum) were taken from participants between April 6th and April 14th. In case of proven SARS-CoV-2 infection, the participant and the health authorities were informed immediately. This study was carried out in accordance with the Declaration of Helsinki and the guidelines of the International Conference for Harmonization for Good Clinical Practice. The study was approved by the local Ethics Committee at the University Hospital Frankfurt. Informed consent was waived due to the retrospective character of the study. All participants have given their written consent. 21.5% of the participants were women and 78.5% were men, all aged between 18 and 65 years ( Table 1 ). The distribution of the participants' places of residence is shown in Figure 1 . The participants showed All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Amplification was done in a multiplex CE certified assay in the ORF region as well as in the E-gene. All samples were tested in accordance to the instruction for use from the manufacturer (Roche Diagnostics, Mannheim, Germany). Among 1,000 individuals screened, we found one person positive for SARS-CoV-2. This person had the nasopharyngeal swab taken on April 9th. The finding was reported to the volunteer on April 12th. No symptoms were recorded at this time. On April 14th a sudden loss of taste in the early morning was described that persisted for 24 hours. On April 15th to 16th a mild headache was reported, from April 17th on there were no further symptoms recorded. Serological assays have been published that are based on the recombinant SARS-CoV-2 proteins N, S or truncated versions of the S protein (S1) for the detection of antibodies against SARS-CoV-2 [1] [2] [3] [4] [5] [6] [7] . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 25, 2020. . https://doi.org/10.1101/2020.05.20.20107730 doi: medRxiv preprint Although the results vary, the studies agree that the S1-based tests are most specific for SARS-CoV-2 and have the least cross-reactions with other coronaviruses [4, 5] . For the development of an in-house ELISA for the detection of anti-SARS-CoV-2 antibodies we used 100 ng recombinant SARS-CoV-2 S1 per well (Sino Biological, 40591-V08H) in combination with an HRP-coupled anti-human IgG All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 25, 2020. . https://doi.org/10.1101/2020.05.20.20107730 doi: medRxiv preprint Among the 29 reactive sera, SARS-CoV-2 neutralising antibodies were detected in five sera ( Figure 3A ). These sera were also reactive to the spike protein at the surface of S1 expressing cells by native immunofluorescence analysis (nIFA as described in Krähling et al., 2016 [10] , with the exception that a pCAGGS-SARS-CoV-2 spike expression plasmid was used) ( Figure 3B ). The remaining 24 sera were not reactive in either assay. We conclude that five out of 1,000 individuals tested developed SARS-CoV-2-specific antibodies. The remaining 24 individuals are likely to have cross-reactive antibodies from other human coronavirus infections. The five sera that were considered truly positive were reactive in the nIFA and in the neutralization test (NT), which indicated that it is possible to confirm the ELISA results with nIFA. This is an important result for laboratories that do not have access to a BSL3 facility. Two of the five seropositive volunteers stated that they underwent a laboratory-confirmed SARS-CoV-2 infection (PCR-based). The other three subjects had not been aware of the infection. The first subject of this group had no symptoms in the past months and no known contact with a COVID-19 patient. The second subject had common cold symptoms 7 weeks before the start of the study, which was assumed to be caused by pollen allergy. This individual went to work regularly in overall healthy condition. The third subject had contact with a colleague who had subsequently tested positive for SARS-CoV-2. As a precaution he stayed at home for two weeks. At no time did he experienced any symptoms. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 25, 2020. . https://doi.org/10.1101/2020.05.20.20107730 doi: medRxiv preprint The 29 serum samples reactive in the SARS-CoV-2 ELISA were further analyzed using a SARS-CoV (2003) neutralisation test (FFM-1 isolate, GenBank accession number AY310120). The assay was performed as described for SARS-CoV-2 with the exception that the CPE was evaluated at day three post infection. Four out of five sera that bound the SARS-CoV-2 S1 protein and neutralised the SARS-CoV-2 virus also neutralised SARS-CoV. In contrast to published results [11], we do see a weak crossneutralization of SARS-CoV by SARS-CoV-2-specific antisera. This can be due to the use of different test systems. Ou and colleagues used pseudotyped VSV for their neutralization tests while we used wildtype SARS-CoV-2 and SARS-CoV. Most of the officially recorded COVID-19 cases showed symptoms or had relevant contact with positively tested index patients. In contrast, among the 998 tested employees with unknown SARS-CoV-2 status, we found four who did not know they were acutely or previously infected with SARS-CoV-2 (95% credibility interval (CrI): [0.163%; 1.022%]; Bayesian analysis using a flat prior [12] ). This result indicated the rate of unreported to reported SARS-CoV-2 cases in this study group was 2, a value which was unexpectedly low when compared with other respiratory diseases such as influenza (underreporting factor 79, USA, flu pandemic 2009, [13] ). One limitation of our study is that the sample was not random because only volunteers were included, so the sample may not be representative. A SARS-CoV-2 surveillance study, performed in Heinsberg, North Rhine Westphalia, Germany, reported 15.5% SARS-CoV-2-infected individuals in a group of 919 [14] . The site of this study was a hot spot of SARS-CoV-2 transmission. The different SARS-CoV-2 prevalences (0.6% Frankfurt vs 15.5% Heinsberg) reflect the inhomogeneous pattern of the SARS-CoV-2 pandemic in Germany. To provide a reliable estimation of the average rate of SARS-CoV-2 infections in the general population in Hesse or Germany it is therefore mandatory to perform studies across the entire country and to combine these data. It will be also necessary to perform follow-up serological studies to determine the further spread of SARS-CoV- All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 25, 2020. . https://doi.org/10.1101/2020.05.20.20107730 doi: medRxiv preprint Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19) Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 Antibody Detection and Dynamic Characteristics in Patients with COVID-19 Evaluation of Nucleocapsid and Spike Protein-based ELISAs for detecting antibodies against SARS-CoV-2 SARS-CoV-2 specific antibody responses in COVID-19 patients SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup Anti-SARS-CoV-2 virus antibody levels in convalescent plasma of six donors who have recovered from COVID-19 Serological and molecular findings during SARS-CoV-2 infection: the first case study in Finland Kinetics of SARS-CoV-2 specific IgM and IgG responses in COVID-19 patients Development of an antibody capture ELISA using inactivated Ebola Zaire Makona virus No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV Bayesian credible intervals for binomial proportions in a single patient trial Estimates of the prevalence of pandemic (H1N1) 2009, United States Infection fatality rate of SARS-CoV-2 infection in a German community with a super-spreading event All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted May 25, 2020.