key: cord-1003628-762k3xko authors: Snoeck, C. J.; Vaillant, M.; Abdelrahman, T.; Satagopam, V. P.; Turner, J. D.; Beaumont, K.; Gomes, C. P. C.; Fritz, J. V.; Schröder, V. E.; Kaysen, A.; Pavelka, L.; Stute, L.; Ramos Meyers, G.; Pauly, L.; Hansen, M.; Pauly, C.; Aguayo, G. A.; Perquin, M.; Hanff, A.-M.; Ghosh, S.; Gantenbein, M.; Huiart, L.; Ollert, M.; Krüger, R. title: Prevalence of SARS-CoV-2 infection in the Luxembourgish population: the CON-VINCE study. date: 2020-05-18 journal: nan DOI: 10.1101/2020.05.11.20092916 sha: f92f53af769d35dd045e62f7e96894253d6a58e1 doc_id: 1003628 cord_uid: 762k3xko BACKGROUND: After the World Health Organization declared the outbreak of coronavirus disease to be a public health emergency of international concern on January 30, 2020, the first SARS-CoV-2 infection was detected in Luxembourg on February 29, 2020. Representative population-based data, including asymptomatic individuals for assessing the viral spread and immune response were, however, lacking worldwide. METHODS: Using a panel-based method, we implemented a representative sample of the Luxembourgish population based on age, gender and residency for testing for SARS-CoV-2 infection and antibody status in order to define prevalence irrespective of clinical symptoms. Participants were contacted via email to fill an online questionnaire before biosampling at local laboratories. All participants provided information related to clinical symptoms, epidemiology, socioeconomic and psychological assessments and underwent biosampling, rRT-PCR testing and serology for SARS-CoV-2. RESULTS: We included a total of 1862 individuals in our representative sample of the general Luxembourgish population. Of these, 5 individuals had a current positive result for infection with SARS-CoV-2 based on rRT-PCR. Four of these individuals were oligosymptomatic and one was asymptomatic. Overall we found a positive IgG antibody status in 35 individuals (1.97%), of which 11 reported to be tested positive by rRT-PCR for SARS-CoV-2 previously and showed in addition their IgG positive status also a positive status for IgA. Our data indicate a prevalence of 0.3% for active SARS-CoV-2 infection and an infection rate of 2.15% in the Luxembourgish population between 18 and 79 years of age. CONCLUSIONS: Luxembourgish residents show a low rate of acute infections after 7 weeks of confinement and present with an antibody profile indicative of a more recent immune response to SARS-CoV-2. All infected individuals were oligo- or asymptomatic. Bi-weekly follow-up visits over the next 2 months will inform about the viral spread by a- and oligosymptomatic carriers and the individual changes in the immune profile. Introduction socioeconomic factors were used to better understand the nature, dynamics of spread, and transmission along with the prevalence of the virus in the population. This study is a nation-wide, observational study aiming to define the prevalence of SARS-CoV-2 infections in the Luxembourgish population. Here we present the baseline assessment of the CON-VINCE cohort. To capture the dynamics and impact of the virus spreading over time, the cohort will be follow-up over 12 months. An intensive data and sample collection will be performed every 2 weeks for the first 2 months (5 times in total) with a final follow-up 1 year after the participants' inclusion in the study. At each collection time-point, blood, nasal and oropharyngeal swabs are collected, and participants also fill in questionnaires on epidemiological and clinical data as well as socioeconomic and psychological well-being. The provision of a stool sample at each biosampling time point is optional for all participants. To allow for data harmonization and international collaboration the overall data set includes questions recommended by the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC), hosted by the Centre for Tropical Medicine and Global Health at the University of Oxford (WHO), and the Weizmann Institute (Rossman et al., 2020) adapted to the Luxembourgish environment (Börsch-Supan et al., 2013; Börsch-Supran, 2019) . The study design (Figure 1 ) accounts for the need to recruit a representative sample of the Luxembourgish population (>18 years old) within a short time frame in the context of the already existing confinement measures. All participants were tested for SARS-CoV-2 by rRT-PCR. Additionally, serological testing for virus-specific antibodies (IgA and IgG against SARS-CoV-2) was performed. Participants who were either asymptomatic or oligosymptomatic, from the clinical point of view, are being followed up longitudinally regardless of the SARS-CoV-2 positive or negative status. Symptomatic individuals positive for SARS-CoV-2 with COVID-19 disease contribute to the baseline assessment but are not followed-up. The baseline questionnaire captured demographic data, medical history and behavioral and psychological data. Demographic data included age, gender, origin, residential areas as well as marital status, number of children, the household composition, and the age of the household members. As socio-economic-status is intimately linked to the incidence and severity of respiratory tract infections, we obtained information on educational level, professional background, current employment status, income, and the house-ownership. Subjects provided information on their medical history, including cardiac, hepatic, metabolic, pulmonary, neurological, hematological and oncological comorbidities, and gynecological history. Prior history of allergies and smoking were recorded. A self-reported description of chronic medication taken regularly was mandatory. COVID-19-related data included whether the participants have already been tested for the SARS-CoV-2, indicating the date and the result of the test. Additionally, we invited subjects to document if they had travelled to an area with confirmed SARS-CoV-2 infections during the 14 days prior to their participation in the study. Environmental conditions of the household were obtained (e.g., the possibility of quarantine of one household member, etc.). To assess psychological and behavioural factors or changes during the pandemic, we asked participants to quantify physical activity, the frequency of leaving the house, alcohol consumption, screen time, as well as social contact through technological devices. Their compliance with the recommendations and restrictions issued by the Luxembourgish government during the pandemic was measured by a series of questions compiled from the WHO and Luxembourg Health Directorate guidelines (WHO 2014 , Luxembourg Health Directorate, 2020 . Depressive symptoms are and further will be assessed in the multiple follow-up questionnaires using the Center for Epidemiologic Studies Depression Scale (CES-D Scale) (Radloff, 1977) . Presence and severity of anxiety is measured in the multiple follow-up questionnaires using the seven items Generalized Anxiety Disorder Assessment (GAD-7) (Spitzer et al., 2006) . Participants' perception of their psychosocial stress level was assessed using the 4-item Perceived Stress Scale (PSS-4) on a 5-point Likert scale (Cohen et al., 1983) . Social isolation and loneliness were assessed with the 3-item UCLA Loneliness Scale (short version) (Hughes et al., 2004) . The Brief Resilience Scale (BRS) (Smith et al., 2008) measures resilience or the ability to recover from a stressful period or event using 6 items, scored on a 5-point Likert scale. Five personality traits (Extraversion, Agreeableness, Conscientiousness, Emotional Stability and Openness) are assessed using the Big Five Inventory-10 (BFI-10) (Rammstedt and John, 2007) . The bi-weekly follow-up questionnaires will cover participants' present health and psychological status. Current health status will focus on signs and symptoms associated with SARS-CoV-2 during the intervening period and medication (e.g., paracetamol, cough medicine, NSAIDs) for symptom relief. Psychological data that will be collected during the follow-up questionnaires include CES-D, GAD-7, PSS-4 and UCLA loneliness scale. BRS and BFI-10 are administered again during the yearly follow-up. As social adversity has been linked to respiratory tract infections (Elwenspoek et al., 2017) the final questionnaire includes the 28-item Childhood Trauma Questionnaire (Bernstein et al., 2003) , to retrospectively assess distress during childhood together with a questionnaire covering the principal psychosocial stressors in adulthood such as divorce, job loss or the death of a family member (Turner at al., 2020) . The questionnaires, as well as the questionnaire schedule, are included in Supplementary Material 1. The study was approved by the national research ethics committee (Comité National d'Ethique de Recherche, CNER), under reference 202004/01, and by the Luxembourgish Ministry of Health under reference 831x6ce0d. The study has been submitted for registration on ClinicalTrials.gov (NCT04379297). The CON-VINCE study was launched on April 15, and participants randomly selected from a representative panel, which consented to the study and fulfilled the inclusion criteria, were enrolled from April 15 until May 5, 2020. Participants filled the online questionnaire and accomplished biosampling in approved diagnostic laboratories spread all over the country. Participants vulnerable to SARS-CoV-2 infection were sampled at home. A total of 1862 participants successfully completed the questionnaire and were biosampled. Given the rapidly evolving incidence of infected cases on April 10, when the study protocol of CON-VINCE was established and based on an unknown number of asymptomatic people, a prevalence of 50% of cases was assumed, that would lead to the largest sample size. It would allow estimating any other prevalence figure. Assuming a 95% confidence interval and a precision of 2.5% around the estimate of prevalence, the required sample size was a minimum of 1537 participants. The chosen sampling strategy for the sample to be representative of the general population was to stratify by gender, age categories (10 years from 18 years and above), and electoral districts. To compensate for non-response and potential drop-outs during the study, over 2000 individuals were invited to join the study. According to the National Institute of Statistics and Economic Studies of the Grand Duchy of Luxembourg (Statec), the national population aged 18 years and over was 514,921 at the beginning of the study. This constituted the sampling frame. An equal allocation probability was used (chance for all individuals of the same age category and gender to be selected) proportional to size (of the population) without replacement (the same individual could not be selected twice). The selection probability for unit i (for example 60-69y) in stratum h (for example men) equaled nhZhi, where nh was the sample size for stratum h, and Zhi was the relative size of unit i in stratum h. The relative size equaled Mhi/Mh, which was the ratio of the size measure for unit i in stratum h (Mhi = number of men aged 65-69y) to the total of all size measures for stratum h (Mh = number of men) (SAS Institute Inc, 2009). Due to constraints related to the emerging pandemic, the sample of participants was enrolled through the use of a non-probabilistic web panel (unknown probability to opt-in) of 18,000 members by a survey company to access participants within the sampling plan. A deterministic random bit generator (DRBG) within strata was used to apply the equal allocation probability proportional to size. The first round of biosampling was performed from April 16 onwards at routine medical diagnostic laboratories (BioneXt, Ketterthill and Laboratoires Réunis) throughout Luxembourg using standardised study collection kits containing all the required materials provided by the Integrated Biobank of Luxembourg (IBBL). Collection kits included the mandatory blood samples and nose and throat swabs, as well as an optional stool collection kit (Supplementary Table 1 ). Following collection, blood, nose and throat swabs were maintained at 2-8°C and transported within 24h to the IBBL for processing. Participants collected stool samples at home and sent them in provided transport boxes to the IBBL using the regular postal service. Blood and stool samples were processed in the IBBL. Swab samples were transferred to the Laboratoire National de la Santé (LNS) for rRT-PCR SARS-CoV-2 testing (Supplementary Table 1 ). Serum was transferred at -20℃ to the Department of Infection and Immunity of the Luxembourg Institute of Health (LIH) for serology testing. Leftover swab samples and residual RNA from swab samples were returned to IBBL for aliquoting and storage, within 24h following the initial transfer to LNS. The sampling procedure will be maintained for the follow-up visits. As part of our Case Report Forms (CRFs) (Supplementary Material 1) was based on ISARIC COVID19 Core CRF (https://isaric.tghn.org/COVID-19-CRF/), our data is aligned with international standards and can be easily harmonized and pooled with other studies facilitating cross-study analysis. Encrypted pseudonymised raw data has been deposited in the Luxembourg Centre for Systems Biomedicine (LCSB) secure repository. Upon arrival, all pseudonymised questionnaire and clinical data is curated and mapped to associated rRT-PCR and serology data through each participant's pseudonymised ID with corresponding sample kit ID (subject-samplemapping) and maintained in REDCap (www.project-redcap.org), personal information was maintained separately in a proprietary secured server (SMASCH). REDCap provides a complete audit trail for study data (https://www.project-redcap.org). This integrated, curated and processed data is available through an access-controlled study-specific data integration and analysis platform (ADA). The ELIXIR-Luxembourg node (ELIXIR-LU http://elixir-luxembourg.org) will provide long-term hosting, ensuring the sustainability of CON-VINCE data. Data flows are highlighted in Figure 2 ). Automated RNA extraction was performed using a STARMag 96 x 4 Universal Cartridge Kit (Seegene) for all swabs. SARS-CoV-2 detection was carried out using the Allplex 2019 n-CoV Assay (Seegene) according to the manufacturer's instructions. The Allplex 2019 n-CoV assay amplifies specific regions in the RdRP, N genes (specific SARS-CoV-2 detection) and E gene (pan-Sarbecovirus detection). Inconclusive results, i.e. samples where only the RdRP or the N gene was amplified, a second manual RNA extraction using the QIAamp viral RNA minikit (Qiagen, Venlo, the Netherlands) followed by duplicate RNA testing with in house assays including the E gene rRT-PCR , N gene (N1 target) rRT-PCR and human RNAse P rRT-PCR as sample quality control (CDC), using TaqPath 1-Step RT-qPCR Master Mix (Life Technologies, Merelbeke, Belgium). Viral RNA from BetaCoV/Germany/BavPat1/2020 strain (Ref 026N-03889), kindly provided by the Charité-Universitätsmedizin Berlin through the European Virus Archive Global platform, was used as positive control. Inconclusive samples were also screened with the FTD SARS-CoV-2 assay (Fast Track Diagnostics, Eschsur-Alzette, Luxembourg), which detects N and ORF1ab genes of SARS-CoV-2. As outlined in Figure 3 , all samples that were only positive for N gene in the original rRT-PCR, were considered positive when a Ct value < 40 was obtained for at least one replicate for 2 viral genes in 2 different second-round rRT-PCRs on the same sample. Anti-SARS-CoV-2 IgA and IgG were determined by CE-labelled enzyme-linked immunosorbent assay (ELISA) kits (most recent versions of Euroimmun Anti-SARS-CoV-2 ELISA IgA and Euroimmun Anti-SARS-CoV-2 ELISA IgG) according to the manufacturer's instructions and as described by others (Streeck et al., 2020) . The optical density (OD) was measured at 450 nm from which background OD measured at 650 nm was deducted. OD ratios were calculated by dividing the resulting OD by the OD of the calibrator, which is included in the kit. In house quality controls, prepared to give an expected OD ratio of approximately three times the threshold for positivity, were included in all assays. As per the kit recommendations, samples with OD ratios <0.8 were considered negative, OD ratios ≥1.1 were considered positive and samples with intermediate OD ratios (>0.8, <1.1) were judged borderline positive. A cohort of anonymised archived sera collected from adults during the two winter seasons (cohort A, 2018, n=92; 2019, n=93) prior to the start of the COVID-19 pandemic were tested by both IgA and IgG ELISAs to assess their specificity. All sera had been collected for other purposes than respiratory disease diagnosis. A second cohort of anonymized sera (cohort B, 2020, n=37) was collected at the Centre Hospitalier de Luxembourg (CHL) from patients with documented COVID-19 disease and positive rRT-PCR results. The delay between onset of symptoms and blood drawing ranged from 1 to 26 days. Those sera contributed to evaluating the ELISA sensitivity. All sera with intermediate results were considered positive when calculating specificity and sensitivity. Prevalence of the SARS-CoV-2 RNA infection was measured by rRT-PCR. IgA and IgG serology was also translated into seroprevalence. The infection rate was evaluated through previously reported and current rRT-PCR and IgG positivity. Prevalence estimates were calculated by accounting for the design of the study, namely weights from the sampling strategy as defined by the stratification variables (gender, 10 years age categories and electoral district). A representativeness evaluation was carried out to verify that the proportions estimated from the sample can be extrapolated to the general population. 95% confidence intervals (95%CI) were given. Sampling proportions were calculated for each strata of the sample of participants enrolled in the study as well as in the general population. The ratio of these proportions provided the weights for post stratification. In the sense of sensitivity analyses, sampling weights were also calculated for "cantons" which is a more granular strata than electoral district. The formula for infection rate was as follows: past or current positive PCR or IgG positive or intermediate divided by the total sample population (N=1835). Prevalences were calculated with SAS v9.4 (SAS institute, Cary, NC, USA) except the IR and IFR where R was used with the svycipro function (Survey package) that calculates 95% confidence intervals for proportions with logit » method. Figures were prepared in R studio (R Core Team, 2019) using the tidyverse (Wickham et al. 2019) , ggbeeswarm (Clarke and Sherril-Mix, 2017 ) and ggpubr (Kassambara, 2020) packages. The recruitment of Luxembourg residents for the CON-VINCE study started on April 15 and was concluded on May 5 after the inclusion of 1862 individuals, thereby exceeding the minimum of 1537 participants calculated for assessing prevalence. All individuals that accomplished the baseline questionnaire online underwent biosampling. Viral rRT-PCR was performed in 1842 participants and SARS-CoV-2 specific serology for IgA and IgG was performed in 1820 individuals. The basic epidemiological features of the study participants, such as gender, age, education, number of persons sharing the same household, and residency within the Luxembourg territory are described in Table 1 . Two serum cohorts were used to assess the specificity and sensitivity of both IgA and IgG ELISAs. In specificity cohort A, 20/185 (10.8%) and 4/185 (2.2%) sera reacted in IgA and IgG ELISAs (positive and intermediate; Figure 4 ), leading to a specificity of 89.2% and 97.8% respectively. Only one serum was IgA and IgG positive, providing an increased specificity of 99.5% when combining both results. Cohort B consisting of hospitalized COVID-19 patients was used to estimate assay sensitivity in relation to the time delay after symptom onset ( Table 2) . IgA and IgG ELISA sensitivity reached 92.9% and 85.7% at 15 days (d15) after symptom onset, while the combination of IgA and IgG results provided a sensitivity of 85.7% ( Table 2) . The presence of anti-SARS-CoV-2 IgA and IgG was assessed in all sera using the CE-labeled Euroimmun assays. Overall, 201/1820 (11.0%), and 35/1820 (1.9%) participants had IgA or IgG antibody levels above the threshold considered for positivity in our study (OD ratio ≥0.8), respectively. Among those, 30 (1.6%) participants were positive for both IgA and IgG (Table 3; Figure 5 ). A positive correlation between IgA and IgG OD ratios was observed (all data, Spearman correlation coefficient r=0.362, p<0.001; IgA positive and IgG positive only, r=0.855, p<0.001). rRT-PCR screening at study baseline A total of 1842 upper respiratory tract swabs were analyzed by Allplex 2019 n-CoV Assay. Six (0.3%) gave inconclusive results, i.e. a positive result was only detected by N gene rRT-PCR. Low levels of SARS-CoV-2 viral RNA, as shown by the high Ct values (Table 4) , were confirmed for 5/6 participants after retesting by additional rRT-PCRs (Figure 3) . Despite the very low viral RNA concentrations, the repeated viral RNA detection combined with the serological response observed strongly suggested that these five participants had been infected with SARS-CoV-2. Apparently, they were in the phase of clearing the viral infection and mounting an antibody response. This is further evidenced for participant 3, who already tested positive 25 days prior to study enrolment. All participants were oligo-or asymptomatic, 2 participants had contact with a COVID-19 household member while one participant had traveled in the last 14 days ( Table 6) . rRT-PCR screening prior to enrolment A total of 138/1862 (7.4%) participants had been tested for SARS-CoV-2 by rRT-PCR prior to study enrolment, and 11 of these 138 (8.0%) tested positive. All previously rRT-PCR positive participants were IgA and IgG seropositive at the baseline time point of our study. Conversely, 16/126 (12.7%) and 5/126 (4.0%) previously rRT-PCR negative participants had tested positive for IgA or IgG, respectively. During the period between the initial (March 16-April 3) and baseline (April 15-April 29) testing (mean interval between tests: 26.9 days, range: 13-37 days), viral clearance, shown by a negative rRT-PCR at baseline, occurred in 10/11 (90.9%), while a single participant remained positive at baseline, 25 days after initial testing (see above). Among all IgG positive participants (n=35), only one participant reported travelling within the previous 14 days and three reported having contacts outside the household (confinement measures were already in place since March 16, 2020). Seven participants were working on-site while 13 were working from home. Thirteen (37.1%) participants reported having contact with confirmed or suspected COVID-19 cases, most of the time (11/13, 84.5%), this person being household members ( Table 6) . A wide range of clinical symptoms were reported for the last 14 days prior to enrolment by IgG positive individuals. Symptoms ranged from none or very mild (≤2 symptoms in 19 participants) to more substantial symptoms, including shortness of breath, chest and abdominal pain. The number of symptoms reported tended to be higher in participants with a previous rRT-PCR positive diagnostic (range 0-13, average 4.5) than in participants with no or a negative rRT-PCR result (range 0-9, average 2.5; Table 6 ). The number of symptoms reported by participants with comorbidities (n=9; average 2.3) tended to be lower than in participants with no reported comorbidities (n=26; average 3.5). For the prevalence evaluation, two out of 1842 participants with rRT-PCR results were excluded because the gender was not defined. Participants aged > 79 years were also excluded (n=12) because representativity for the overall population was not reached for this age group. The target population for prevalence evaluation was therefore defined as people between age 18 years until age 79 years. The number of individuals with rRT-PCR and serology results available was 1830 and 1807, respectively with 4 participants negative in serology with no PCR results. The sampling frame was used to weight the number of individuals in each cell defined by the three stratification variables. Weights were calculated by dividing each cell count from the target population by the one from the sample. It was therefore used in post-stratification. The evaluations of prevalence used the weights to extrapolate to the target population and evaluate the 95% confidence intervals. Table 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 18, 2020. . As a sensitivity analysis, weights were recalculated using Luxembourg's regional cantons rather than electoral districts, a more granular geographic representation. An electoral district is composed of 2 to 5 cantons. Participants were randomly selected in the district, but as we also obtained the canton information, it was relevant to have an estimate of how the prevalence could be biased by potential clustering within districts. Using the more granular canton approach, the time prevalence of SARS-CoV-2-rRT-PCR was 0.32% (95%CI=[0.02;0.63]) which was an estimate very close to that obtained at the district level. A similar observation could be made for the other estimated prevalence (Supplementary Table 2 ). The present study provides first prevalence data on the spread of SARS-CoV-2 in the population of Luxembourg using a panel-based approach. The weighted prevalence of SARS-CoV2 carriers based on rRT-PCR reveals a low prevalence of 0.30%, which may be due to the confinement measures in place for more than 7 weeks, including social distancing for the overall population and self-isolation and quarantine. Our data suggest that between April 16 and May 5 there were 1,449 adults in Luxembourg that were oligo-or asymptomatic carriers of the SARS-CoV-2. Similarly, low results from population screenings in Iceland were recently reported with 0.8% virus-positive individuals in an open-invitation screening and 0.6% in a random-population screening identified (Gudbjartsson et al., 2020) . The relevance of assessing the contribution of asymptomatic and oligosymptomatic individuals to the dynamics of the pandemic is increasingly recognized and also motivated a study at the National Institute of Health (USA) using a similar population-based approach where participants fill questionnaires during a virtual clinical visit and are biosampled for assessing seropositivity (NCT04334954). Of the five SARS-CoV-2 positive participants only one reported that he had travelled to a COVID-19 risk area. Two positive participants reported prior contact with a COVID-19 diseased household member. The source of infection for the other 2 SARS-CoV-2 positive participants is not clear, but most probably within Luxembourg, as travels were limited given the confinement measures in place. For the presence of infected individuals within a household, we observed the highest percentage in households with 2 members and the lowest percentage in households with 5 or more individuals (Suppl. Fig. 1 ). This is in line with the observation in a recent German study that showed a relatively moderate increase of the secondary infection risk dependent on the household cluster size (Streeck et al., 2020) . Overall participants tested positive for an active infection of SARS-CoV-2 by rRT-PCR, were oligoor asymptomatic and presented with fewer symptoms (mean of 1.6 symptoms out of 22; SD=1.14) than individuals with negative rRT-PCR results (mean of 2.3 symptoms out of 22; SD=2.43). This 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 18, 2020. . underscores the relevance of infected individuals that do not display typical COVID-19 signs and symptoms for the viral spread during the pandemic. In line with previous reports, we see males overrepresented in the group of SARS-CoV-2 positive participants (4 males versus 1 female) (Gudbjartsson et al., 2020; Guan et al., 2020) . As we targeted a population >18 years old, we cannot make any direct statement on the contribution of children to the viral spread. All of the data was collected through online questionnaires. In highly connected countries such as Luxembourg, approximately 97% of the population accesses and regularly uses the internet (International Telecommunications Union, 2019). Previously, self-reported online questionnaires have been shown to be a reliable method of accessing the clinical evolution of disease (Davies 2016) , and the lack of face-to-face interaction, flexibility to complete the questionnaires at the participants' convenience, together with the perceived degree of anonymity inherent in such online questionnaires is thought to enhance data accuracy, reducing central coherence and social desirability biases at the cost of participants potentially misinterpreting the questions (Ong and Weis, 2000) . Retrospective assessment was limited to events and symptoms occurring in the previous two weeks to reduce recall bias. The rapidity with which such a self-reporting system that was accessible throughout the population was rolled out was crucially important in the pandemic situation. This rapidity means that reliable data can be provided promptly to researchers during the ongoing pandemic. Furthermore, in such an unpredictable, rapidly changing pandemic situation follow-up questionnaires can be modified as quickly as ethical approval can be obtained. The advantages were, however, counterbalanced by the possibility of survey fatigue with five survey waves in quick succession. The geographical location of Luxembourg and the nature of the study required that several compromises were made. Luxembourg is highly multilingual and successfully recruiting a representative cohort required that study participants were offered the choice of questionnaires in French, German, English and Portuguese. The problems associated with translating questionnaires are well known (Pan and Fond 2014), and to circumvent this, we restricted the study to questionnaires that had previously been experimentally validated in all languages. As we aimed to detect asymptomatic SARS-CoV-2 carriers the ISARIC case report form was adapted to participants that had limited or no symptoms and the Luxembourgish sociodemographic situation. There are pros and cons of using web panels for surveys. We used the services of a private company for immediate access to potential participants from all over the country and based on prior knowledge on age, gender and residency. The sample of participants to the current study was enrolled through the use of a non-probabilistic web panel (unknown probability to opt-in) of 18,000 panel members. Registration to the panel was constituted via invitation during telephone or face to face interviews as well as ad banners, media campaigns and homepage of the company. The sample was randomly drawn from the panel for each stratum defined by the crossing of the 3 stratification variables through a deterministic random bit generator (DRBG) within strata. Individuals living across the borders were excluded as we aimed at a sample of Luxembourgish residents. The advantages of using such a panel was the simplification of the logistics to implement the study in a short time frame in order to capture the prevalence of the infection (Figure 6 ) while the decreasing slope of the epidemics has already started. Another aspect to consider is that no hospitalised patient was included in our sample as they were not reachable through the panel. However, online surveys may be biased samples because the respondents are self-selected whereby individuals who have strong opinions, are overrepresented, and individuals that are indifferent or apathetic are less likely to opt-in or respond. Nevertheless, we tried to correct the selection bias and under representation by first stratifying the sampling on gender, age and residency at the sampling level and second, by post stratification at the analyses level. Overall, our study captured 6 rRT-PCR positive participants with low viral RNA levels. These may be considered negative in other studies depending on the criteria applied. The combination of retesting by additional rRT-PCRs (Figure 3 ) as well as assessment of IgA and IgG serology, however, strongly suggest that 5 of these 6 study participants were true SARS-CoV-2 positive cases with residual low viral loads and seroconversion. This is also further evidenced by one of the participants who already tested positive 25 days prior to enrolment (participant 3, see Table 4 ). Duration of SARS-CoV-2 viral shedding appears to vary widely between patients. Detection of viral RNA for 2-3 weeks after symptom onset is frequent (Kujawski et al., 2020; Wölfel et al., 2020) , even in seroconverted patients, and extended viral shedding for up to 83 days was reported (Li et al., 2020). One of the study participants (participant 5, Table 4 ) was not confirmed positive after two confirmation rRT-PCR assays and did not show seroconversion until now. This patient might have been recruited at the onset of viral shedding in contrast to the other cases and/or may not mount an immune response as sometimes observed in asymptomatic patients. Longitudinal follow-up sampling as implemented in the design of our study will allow us to clarify the status of this participant at a later stage. The frequency of seasonal hCoV infections in the general population, including adults (Monto et al., 2020), coupled with possible concomitant infections by different hCoV (Heimdal et al., 2019) suggest a limited duration of protection for each hCoV and limited cross-protection between hCoV. Nevertheless, cross-reactivity in ELISA due to past exposure to other hCoV likely partially accounts for the observed lower specificity of the anti-SARS-CoV-2 IgA ELISA used in our study. Notably, cross-reactivity in anti-SARS-CoV-2 IgA and IgG ELISAs has been documented in patients with specific seroconversion against hCoV-OC43, while baseline sera were negative (Okba et al., 2020) . In Germany, the follow up of a birth cohort revealed that 19/25 (76.0%) newborns had seroconverted by 21 months of age (Dijkman et al., 2012) . While the frequency of each of the four seasonal hCoV oscillates between years and regions, hCoV-OC43 (betaCoV) is usually more frequent, in patients with acute respiratory disease, especially compared to hCoV-229E (alphaCoV) (Dijkman et al., 2012; Heimdal et al., 2019; Monto et al., 2020) . Aside from a lower assay specificity, discrepancies between IgA and IgG responses may also arise from sequential immune response. IgA tends to appear earlier than IgG after symptom onset (Guo et al., 2020) , and may explain why some participants have detectable levels of IgA but no 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 18, 2020. . In this study, intermediate IgA and IgG antibody levels were assimilated to a positive serological response, as done previously (Streeck et al., 2020) . Although this approach decreases specificity, the current study design allows for a careful monitoring of seroconversion trends and antibody levels over time during subsequent biosampling of study participants, which will allow us a better interpretation of evolving antibody responses. Additional immunoassays including alternative antigens or antigen fragments as well as neutralization assays will complement the panel of tests that will be performed at a later stage of the CON-VINCE study. These additional immunoprofiling data will provide a more refined assessment and evaluation of the ELISA assay results obtained at baseline level of our study. The longitudinal design of our study with bi-weekly follow-ups will allow us to assess the dynamics of the pandemic in our study population along with the gradual easing of the protective measures taking place in Luxembourg as well as other European countries. This may inform about the impact of oligo-and asymptomatic carriers on the viral spread during the upcoming months. Moreover, we will be able to study immune responses via continuous serological testing and may provide information on the risk of re-infections, as knowledge about SARS-CoV-2 immunity is still limited. Elwenspoek Statec. (2020). 626,000 inhabitants as of January 1, Figure 1 : Study design and testing. A representative sample of the Luxembourgish population was invited to join the study. Following the completion of an online questionnaire and biosampling, rRT-PCR and serology were performed. Asymptomatic and oligosymptomatic participants (either positive or negative for the virus) were followed up on a bi-weekly basis for a total of 4 times. A final follow-up will take place after one year. Data and sample flow in CON-VINCE cohort. Study participants are recruited by a survey company (TNS-Ilres) based on a large representative panel of residents of Luxembourg. Participant personal data is securely collected and can only be accessed by the clinical study team. Each participant's personal record is assigned with a pseudonym. All data is collected by a protected web-based interface and transferred along with the pseudonym to the Data and Computing Platform hosted at the LCSB in a secure data center. Biosamples are collected at different sample collection hubs across Luxembourg and shipped to IBBL for sample processing and biobanking. Biosamples are analysed at the Laboratoire National de Santé (LNS) and the Luxembourg Institute of Health (LIH) and the analysis results together with biosample annotations and barcodes are recorded in REDCap. Within the Data and Computing Platform, the pseudonymized data and results from biological analyses from REDCap is accessed by the Data Integration and Analysis Platform (Ada) via an Application Programming Interface and integrated results with clinical data made available through its secure and access-controlled web-application. 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 18, 2020 . . https://doi.org/10.1101 /2020 Figure 3: Algorithm for swab testing by rRT-PCR and decision tree 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 18, 2020. . (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 18, 2020 . . https://doi.org/10.1101 /2020 (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 18, 2020. . (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 18, 2020. . a All inconclusive samples were negative in Allplex 2019-nCoV Assay E gene and RdRp gene targets, but positive in N gene. b Confirmatory testing was done by in house generic rRT-PCR assays for the E gene and N gene targets and in addition by the FTD SARS-CoV-2 assay using N gene and ORF1ab targets. (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 18, 2020. . 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 18, 2020 . . https://doi.org/10.1101 /2020 Supplementary 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 18, 2020 . . https://doi.org/10.1101 /2020 Supplementary Material 1 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 18, 2020 . . https://doi.org/10.1101 /2020 (full extended part with defined minimal data set) 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 18, 2020 . . https://doi.org/10.1101 /2020 Infection by Coronavirus with severe disease course requiring a hospital admission prior to the inclusion to the study. Presence of fever and respiratory distress/cough at the time of inclusion not attributable to other known chronic disease. Explanatory note for participant: The virus called SARS-CoV-2 (further indicated as Coronavirus) is the cause of the current pandemic disease known as COVID-19. It is a rapidly spreading virus affecting mainly the respiratory tract having up to 80% an asymptomatic course (meaning without any symptoms) or very mild upperrespiratory disease with runny nose or pink eye. However, in a fraction of patients, the disease evolves to fever and cough with or without respiratory distress and in some cases, a hospitalisation with therapy by inhaled oxygen and further medical support is needed. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Have you been in contact with suspected COVID-19 positive samples? 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 18, 2020 . . https://doi.org/10.1101 /2020 (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 18, 2020 . . https://doi.org/10.1101 /2020 (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 18, 2020 . . https://doi.org/10.1101 /2020 Filter A person at risk is defined either by age (over 60) and/or being affected by coronary heart disease/ hypertension/ diabetes/ chronic pulmonary disease and/ or having compromised immune system by an immuno-suppressive therapy or due to an immuno-compromising disease. ☐ Yes ☐ No ☐ Unknown 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 18, 2020 . . https://doi.org/10.1101 /2020 Are you familiar with the recommendations and restrictions issued by the government in Luxembourg? To what extent do you agree with the following statements? Strongly disagree Disagree Neutral Agree Strongly agree I have tried my best to follow these recommendations I followed the general hygiene recommendations (handwashing, sneezing in elbow/fresh tissue, avoiding to touch my face) I followed the general social distancing recommendations (avoiding handshaking, keeping 2m distance, staying home, avoid traveling) How often did you leave the house during the last week? Presumed Asymptomatic Carrier Transmission of COVID-19 Development and validation of a brief screening version of the Childhood Trauma Questionnaire Measuring digital development: Facts & figures Data Resource Profile: The Survey of Health, Ageing and Retirement in Europe (SHARE) Survey of Health, Ageing and Retirement in Europe (SHARE) Wave 6 ggbeeswarm: Categorical Scatter (Violin Point) Plots v 0.6 A global measure of perceived stress Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR Insights into rare diseases from social media surveys Clinical Characteristics of Coronavirus Disease 2019 in China Spread of SARS-CoV-2 in the Icelandic Population Human coronavirus in hospitalized children with respiratory tract infections: A 9-year population-based study from Norway Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet A short scale for measuring loneliness in large surveys: Results from two population-based studies Infection fatality rate of SARS-CoV-2 infection in a German community with a super-spreading event Twin Research in the Post-Genomic Era: Dissecting the Pathophysiological E ff ects of Adversity and the Social Environment Welcome to the Tidyverse Virological assessment of hospitalized patients with COVID-2019 Infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care Coronavirus (COVID-19). https://covid19.who.int/ World Health Organization (WHO). (2020b) Please indicate your current height (in cm)? Please specify: ______________________________ Please indicate your current weight We would like to give special thanks to all participants of the CON-VINCE study. Additionally, we are very grateful for the financial support by the Fonds National de la Recherche (FNR) and the André Losch Foundation, which enabled us to carry out the project. The funders had no role in the design and conduct of the study, nor in the decision to prepare and submit the manuscript for publication.We would like to thank the Research Luxembourg COVID-19 Task Force (Frank Glod, Paul Wilmes, Lars Geffers, Jasmin Schulz, Henri-Cauchie, Ulf Nehrbass) and Rudi Balling for their overall support of the CON-VINCE study. Yes (