Health
Prevalence of SARS-CoV-2 in Spain (ENE-COVID): a nationwide, population-based seroepidemiological study
Summary
Background
Spain is one of the European countries most affected by the COVID-19 pandemic. Serological surveys are a valuable tool to assess the extent of the epidemic, given the existence of asymptomatic cases and little access to diagnostic tests. This nationwide population-based study aims to estimate the seroprevalence of SARS-CoV-2 infection in Spain at national and regional level.
Methods
35 883 households were selected from municipal rolls using two-stage random sampling stratified by province and municipality size, with all residents invited to participate. From April 27 to May 11, 2020, 61 075 participants (75·1% of all contacted individuals within selected households) answered a questionnaire on history of symptoms compatible with COVID-19 and risk factors, received a point-of-care antibody test, and, if agreed, donated a blood sample for additional testing with a chemiluminescent microparticle immunoassay. Prevalences of IgG antibodies were adjusted using sampling weights and post-stratification to allow for differences in non-response rates based on age group, sex, and census-tract income. Using results for both tests, we calculated a seroprevalence range maximising either specificity (positive for both tests) or sensitivity (positive for either test).
Findings
Seroprevalence was 5·0% (95% CI 4·7–5·4) by the point-of-care test and 4·6% (4·3–5·0) by immunoassay, with a specificity–sensitivity range of 3·7% (3·3–4·0; both tests positive) to 6·2% (5·8–6·6; either test positive), with no differences by sex and lower seroprevalence in children younger than 10 years (<3·1% by the point-of-care test). There was substantial geographical variability, with higher prevalence around Madrid (>10%) and lower in coastal areas (<3%). Seroprevalence among 195 participants with positive PCR more than 14 days before the study visit ranged from 87·6% (81·1–92·1; both tests positive) to 91·8% (86·3–95·3; either test positive). In 7273 individuals with anosmia or at least three symptoms, seroprevalence ranged from 15·3% (13·8–16·8) to 19·3% (17·7–21·0). Around a third of seropositive participants were asymptomatic, ranging from 21·9% (19·1–24·9) to 35·8% (33·1–38·5). Only 19·5% (16·3–23·2) of symptomatic participants who were seropositive by both the point-of-care test and immunoassay reported a previous PCR test.
Interpretation
The majority of the Spanish population is seronegative to SARS-CoV-2 infection, even in hotspot areas. Most PCR-confirmed cases have detectable antibodies, but a substantial proportion of people with symptoms compatible with COVID-19 did not have a PCR test and at least a third of infections determined by serology were asymptomatic. These results emphasise the need for maintaining public health measures to avoid a new epidemic wave.
Funding
Spanish Ministry of Health, Institute of Health Carlos III, and Spanish National Health System.
Introduction
With more than 249 000 confirmed cases and more than 28 000 deaths by July 2, Spain remains one of the European countries most severely affected by the ongoing COVID-19 pandemic.
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However, epidemiological surveillance of confirmed COVID-19 cases captures only a proportion of all infections because the clinical manifestations of infection with SARS-CoV-2 range from severe disease, which can lead to death, to asymptomatic infection.
By contrast, a population-based seroepidemiological survey can quantify the proportion of the population that has antibodies against SARS-CoV-2. A seroepidemiological study provides information on the proportion of the population exposed and, if the antibodies are a marker of total or partial immunity, the proportion of the population that remains susceptible to the virus.
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and others are ongoing.
However, many of them are small or based on non-random sampling of participants (eg, focusing on health-care workers or blood donors) and thus cannot provide precise estimates of seroprevalence by age group in the general population. Additionally, some of these studies have used antibody tests with low sensitivity or specificity or have not reported the characteristics of the test.
Evidence before this study
Spain is one of the European countries most affected by the COVID-19 pandemic so far. Seroepidemiological surveys are a useful tool to track the transmission of epidemics, but few have been done for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We searched PubMed and its specific hub LitCovid, OpenAIRE, Embase, and medRxiv and bioRxiv preprint servers up to May 25, 2020, for epidemiological studies using the terms “seroprevalence” or “seroepidemiology” and “SARS-CoV-2” without date or language restrictions. Most serological surveys were fairly small or focused on specific population subgroups. Large population-based studies are required to understand the dynamics of the epidemic.
Added value of this study
This is the first nationwide population-based study that presents seroprevalence estimates of antibodies against SARS-CoV-2 at national and regional levels, exploring the landscape of population immunity in Spain. With more than 61 000 participants, this study provides accurate prevalence figures according to sex, age—from babies to nonagenarians—and selected risk factors. Our findings confirm that at least a third of individuals who have developed antibodies against SARS-CoV-2 were asymptomatic. Additionally, our results indicate that children and adolescents have lower seroprevalence than adults and seroprevalence does not vary by sex. Our study confirms that a high-quality point-of-care test could be a good choice for large seroepidemiological studies. The rapid test used here showed good performance compared with a chemiluminescent microparticle immunoassay. Finally, the use of two different assays allowed us to define seroprevalence ranges alternatively favouring specificity (requiring a positive result for both tests) or sensitivity (positive to either test).
Implications of all the available evidence
The relatively low seroprevalence observed in the context of an intense epidemic in Spain might serve as a reference to other countries. At present, herd immunity is difficult to achieve without accepting the collateral damage of many deaths in the susceptible population and overburdening of health systems. Our results, together with previous evidence, suggest that approximately a third of people with SARS-CoV-2 infection remain asymptomatic, which has important public health implications. Regional seroprevalence data offer valuable information to tailor public health policies against this epidemic.
In April, 2020, the Spanish Ministry of Health and the Institute of Health Carlos III, in collaboration with the health services of the Spanish regions (Autonomous Communities), launched ENE-COVID, a nationwide, population-based, longitudinal seroepidemiological study, to quantify the extent of SARS-CoV-2 circulation throughout the country. The study included more than 61 000 individuals from randomly selected households; was designed to be representative by province, age group, and sex; and used two tests for the determination of SARS-CoV-2 antibodies. Here, we describe the study design and the results of the first wave of the study, conducted between April 27 and May 11, 2020.
Methods
Study design and participants
This study involved the coordination and training of 29 laboratories and 4400 health professionals in 1409 health-care s throughout the Spanish National Health System. A single ad-hoc information system capable of hosting up to 2000 concurrent users was developed and deployed in approximately 2 weeks to allow daily data modification by study staff for 4000 households and 15 000 participants, alongside the coordination and development of uniform, nationwide support for the study procedures.
The study design includes three successive follow-up waves of data collection, with a 1-week break between them. Each wave is scheduled to be completed within 2 weeks. Half of the cohort (12 households per census tract) was randomly assigned to data collection during the first week of each wave and the other half to the second week, so that serum specimens are collected in all participants 2–4 weeks apart. In this Article, we present seroprevalence data from the first wave of the ENE-COVID study, which was conducted from April 27 to May 11, 2020.
Field work was carried out by staff from each of the region’s health services under a common protocol developed by the Institute of Health Carlos III, which also coordinated the training of all personnel via a web platform. Individuals residing in the selected households were contacted by telephone and invited to either go to their primary health-care s or to allow a home visit, where they provided informed consent. Participants answered a questionnaire that included history of symptoms compatible with COVID-19 (ie, fever, chills, severe tiredness, sore throat, cough, shortness of breath, headache, anosmia or ageusia, and nausea, vomiting, or diarrhoea), contact with suspected or confirmed cases, and other risk factors; had a point-of-care rapid test to detect antibodies against SARS-CoV-2; and, optionally, donated a blood sample for subsequent laboratory analysis. The answers to the questionnaire and the result of the point-of-care test were recorded on site in a secure web application, specifically designed for this study by the Ministry of Health. Blood samples were centrifuged, labelled, and stored refrigerated at the primary health-care s, and sent to the laboratory every 2–3 days. Serum samples were analysed at the National Centre for Microbiology (Institute of Health Carlos III) or in one of 28 selected regional microbiology laboratories.
The Institutional Review Board of the Institute of Health Carlos III approved the study (register number PI 39_2020). The Spanish Data Protection Agency was consulted. Written informed consent was obtained from all study participants. Different forms of informed consent were used for adults, teenagers, parents of participant children, and guardians of mentally disabled participants. Witnesses assisted participants who were not able to read any of the four official languages of Spain.
Detection of SARS-CoV-2 antibodies
Two serological tests were done: a point-of-care rapid test applied directly to fingerprick blood, and a chemiluminescent microparticle immunoassay that requires venepuncture for subsequent analysis in laboratory.
which yields results in 10 min. The manufacturer reported sensitivity of 97·2% for IgG and 87·9% for IgM and specificity of 100% for both IgG and IgM, using RT-PCR as the gold standard. A verification study, done by the National Centre for Microbiology as preparation for ENE-COVID, returned a sensitivity of 82·1% for IgG and 69·6% for IgM in fingerprick blood samples and a specificity of 100% for IgG and 99·0% for IgM (appendix p 11). An independent validation study gave similar results.
Due to the lower sensitivity and specificity of IgM, its shorter duration, and the heterogeneity of results observed in initial IgM readings, results for the point-of-care test reported here are based only on IgG.
Again, a verification study by the National Centre for Microbiology showed a sensitivity of 89·7% in serum samples from RT-PCR-positive patients after 14 days from symptom onset and a specificity of 100% with samples obtained before Dec 8, 2019 (appendix p 12).
Statistical analysis
We estimated seroprevalence as the proportion of individuals who had a positive result in the IgG band of the point-of-care test or, in separate analyses, who had a positive result in the immunoassay. In sensitivity analyses restricted to participants with data on both tests, we estimated seroprevalence as the proportions of individuals who had a positive result in both tests (most specific approach) or in either test (most sensitive approach). We used these analyses to provide a seroprevalence range that either favours specificity (requiring a positive test in both assays) or sensitivity (considering positive results in either test). Using the immunoassay as an alloyed reference, we also estimated the relative sensitivity of the point-of-care test as the proportion of individuals with a positive test among those with a positive result in the immunoassay, as well as the relative specificity as the proportion of individuals with a negative test among those with a negative result in the immunoassay.
Different sampling weights were constructed for the point-of-care test and the immunoassay, with trimmed extreme weights (0·2% and 0·6%, respectively) to counter highly influential observations. The weights for the immunoassay were also used for the sensitivity analysis combining both tests.
Due to the complex study design, all statistical analyses accounted for stratification by province and municipality size group, as well as clustering of seropositivity for SARS-CoV-2 by household and census tract, when computing SEs of seroprevalence estimates. Design effects resulted in inflation factors for SEs of the overall seroprevalence estimates with the point-of-care test of 2·00 and of 1·99 for the immunoassay. Finite population corrections were applied because some sampling fractions of census tracts per stratum and households per census tract were not negligible. 95% CIs were calculated using logit-transformed seroprevalence estimates and their SEs, with the usual design-based degrees of freedom equal to the number of first-stage sampling units minus the number of strata, and were back-transformed to the original scale for reporting. Analyses were done using survey commands in Stata (version 16).
Role of the funding source
The funders facilitated data acquisition but had no role in the design, analysis, interpretation, or writing. The first three authors had full access to all the data. The first five authors and the senior author (RY) had final responsibility for the decision to submit for publication.
Results
Figure 1Flow chart of participants in first wave of the ENE-COVID study
*Care-home residents, hospitalised people, people in prisons, nuns and friars in convents, and residents in other collective residences.
Table 1Seroprevalence of SARS-CoV-2 by general characteristics
SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.
Figure 2Seroprevalence of SARS-CoV-2 by province by the point-of-care test and immunoassay
Figure 3Seroprevalence of SARS-CoV-2 by age
Vertical lines represent 95% CIs. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.
Table 2Seroprevalence of SARS-CoV-2 by self-reported clinical characteristics
SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.
Among those with a positive test, the proportion of individuals who reported anosmia or three or more symptoms compatible with COVID-19 was 49·1% (95% CI 46·2–51·9) for the point-of-care test and 54·2% (51·0–57·2) for the immunoassay. The proportion of individuals with a positive test who were asymptomatic was 32·7% (30·2–35·4) and 28·5% (25·6–31·6), respectively, with a specificity–sensitivity range of 21·9% (19·1–24·9; both tests positive) to 35·8% (33·1–38·5; either test positive). Based on the overall seroprevalence range of 3·7% to 6·2% and the above proportions of seropositive individuals who were asymptomatic, it was estimated that between 376 000 and 1 042 000 asymptomatic individuals went undetected in the non-institutionalised Spanish population.
Among those participants with a history of COVID-19-related symptoms who presented antibodies, the proportion of individuals reporting a previous PCR test ranged between 16·4% (95% CI 13·8–19·5; either test positive) and 19·5% (16·3–23·2; both tests positive). Among them, a positive PCR was obtained in 75·1% (66·9–81·8; either test positive) and 78·8% (70·3–85·4; both tests positive), respectively.
Table 3Relative performance of point-of-care test compared with immunoassay for detection of IgG antibodies for SARS-CoV-2
Relative performance is among 51 958 participants with both point-of-care test and immunoassay. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2.
Discussion
The prevalence in hotspot areas such as Madrid is more than five times higher than that observed in low-risk regions such as most provinces along the coasts.
The use of two IgG antibody tests aimed at different SARS-CoV-2 antigens allows us to specify a range of seroprevalence between 3·7% and 6·2%, depending on whether we favour greater specificity (ie, a positive result in both tests), which might be preferred when prevalence is low,
or greater sensitivity (ie, a positive result in either test). These estimates confirm the magnitude of seroprevalence suggested by smaller studies.
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prevalence increased throughout childhood and adolescence, remained fairly stable at older ages when using the point-of-care test, and, when using the immunoassay only, decreased after age 85 years. The lower prevalence in children might be in part related to lower nasal gene expression of angiotensin-converting enzyme 2.
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In Spain, health-care workers comprise 24% of all confirmed COVID-19 cases—a proportion partly explained by greater access to PCR testing—and 9% of hospitalised cases in their age range.
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Both phenomena—asymptomatic cases and partial ascertainment—are relevant here. The proportion of asymptomatic infections reported in different studies varies greatly, ranging from 4% to 41%.
Here, asymptomatic cases represent between 21·9% and 35·8% of all SARS-CoV-2 infections, corresponding to between 376 000 and 1 042 000 asymptomatic infections in the entire non-institutionalised Spanish population. This finding reinforces the importance of rapid identification, study, and isolation of people with confirmed SARS-CoV-2 infection and their contacts to prevent the spread of the epidemic.
Regarding incomplete ascertainment, only between 16% and 20% of symptomatic participants with antibodies against SARS-CoV-2 reported a previous PCR, and it was positive in around 75–79% of them. We are relying on participants’ retrospectively self-reported symptoms, so a certain amount of misclassification cannot be ruled out. Still, these figures suggest that a substantial number of symptomatic patients with COVID-19 did not undergo PCR testing. However, the fact that only 15·3–19·3% of symptomatic participants had antibodies against SARS-CoV-2 suggests that a sizable proportion of suspected cases might have symptoms not caused by this coronavirus.
and the recently reported 99% of antibody response among confirmed COVID-19 cases.
For the few patients who do not develop antibodies against SARS-CoV-2, it is unknown whether they are susceptible to reinfection.
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Prevalence in those participants reporting negative PCR was higher than in those without a PCR test, which might be explained by delayed PCR testing that yields a negative result or by imperfect sensitivity of PCR tests.
It is important to bear in mind that, in a context of low prevalence figures as those found in this Article, false-positive results might be a relevant issue. Even though the S protein and nucleoprotein show less than 30% similarity with endemic betacoronaviruses, a cross-reaction cannot be ruled out.
In this sense, the combination of both tests provides a more conservative estimation of the real figures.
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The point-of-care test also detected IgM antibodies, but the IgM band had lower sensitivity and specificity, might be positive in presence of rheumatoid factor,
and was subject to substantial variability in initial IgM readings.
could account for around 6% of Spaniards older than 75 years. Even though care homes have been a hotspot of infection and death in the country, most Spanish elders reside in households and they are adequately represented in our study. The remarkably high participation across the country, even in the venepuncture-based assay, reflects the keen interest that the Spanish population has in knowing its serological status. Participation rates were a bit lower in less affluent areas, but this was compensated by adjustment for median income in the census tract. We could not explore differences by race, as this information was not available. However, most participants were Spaniards, who are mostly white. Our study only detected IgG antibodies, but the extent of the immunity they provide is unknown at this moment. However, cellular immunity, which was not evaluated here, might also play a role in protecting against SARS-CoV-2 reinfection.
ENE-COVID provides seroprevalence data at a regional level to inform national and local public health policies. It offers a picture of SARS-CoV-2 circulation that can be compared with surveillance data to evaluate differences in diagnostic exhaustiveness. In addition, comparative performance among regions with similar prevalence but different burden in terms of deaths and health-care capacity could help to suggest areas of improvement and highlight unattended needs that should be considered to face a future epidemic wave.
In conclusion, our study provides nationwide and regional estimates of SARS-CoV-2 dissemination in Spain, showing remarkable differences between higher and lower prevalence areas. One in three infections seems to be asymptomatic, while a substantial number of symptomatic cases remained untested. Despite the high impact of COVID-19 in Spain, prevalence estimates remain low and are clearly insufficient to provide herd immunity. This cannot be achieved without accepting the collateral damage of many deaths in the susceptible population and overburdening of health systems. In this situation, social distance measures and efforts to identify and isolate new cases and their contacts are imperative for future epidemic control.
MP, BP-G, and RP-B were responsible for the conception and design of the study. RY and FB are the executive coordinators of the project and led the relationship with regional health services. JO, MP-O, AF-G, and PM-A are responsible for validation studies to choose the serological tests, the coordination of participant microbiological laboratories, and acquisition of laboratory data. JLS, MMa, JFM-M, and JLP are responsible for the study operation, including the coordination of data acquisition and logistics. IC and MMo developed the operational protocols for field work and were responsible for training the involved administrative and health personnel. MP, BP-G, RP-B, NFdL, FR-C, and MAH were in charge of statistical analyses and table and figure design. All remaining authors in the ENE-COVID group contributed to data acquisition, laboratory analyses, and quality control for their respective regions or at national level. The first draft was written by MP, BP-G, RP-B, MAH, and JO. All authors contributed to data interpretation, critically reviewed the first draft, and approved the final version and agreed to be accountable for the work.
We declare no competing interests.
Acknowledgments
This work was supported by the Spanish Ministry of Health, the Institute of Health Carlos III (Ministry of Science and Innovation), and the National Health System, including the Health Services of all Autonomous Communities and autonomous cities: Servicio Andaluz de Salud, Servicio Aragonés de Salud, Servicio de Salud del Principado de Asturias, Servei de Salut Illes Balears, Servicio Canario de la Salud, Servicio Cántabro de Salud, Servicio de Salud de Castilla-La Mancha, Servicio de Salud de Castilla y León, Servei Català de Salut, Conselleria de Sanitat Universal i Salut Pública de la Generalitat Valenciana, Servicio Extremeño de Salud, Servizo Galego de Saúde, Servicio Riojano de Salud, Servicio Madrileño de Salud, Servicio Murciano de Salud, Servicio Navarro de Salud-Osasunbidea and Instituto de Salud Pública y Laboral de Navarra, Servicio Vasco de Salud-Osakidetza, and Instituto de Gestión Sanitaria. The Spanish Institute of Statistics provided the random selection of households and the information required for participants’ contact. We thank all the nurses, general practitioners, administrative personnel, and other health-care workers who collaborated in this study and all participants. This study is the result of the efforts of many professionals and the trust and generosity of more than 61 000 participants who have understood the importance of providing time, information, and samples to learn about the COVID-19 epidemic in Spain.
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