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.
The findings from this nationwide seroprevalence study for SARS-CoV-2 indicate that the prevalence of IgG antibodies against this coronavirus is around 5% in Spain. Because the study was designed to obtain representative data at both national and provincial level, we were able to observe marked regional differences between the centre of Spain and the outskirts that generally match the surveillance data.2 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.
To our knowledge, ENE-COVID is the largest population-based SARS-CoV-2 seroprevalence study in Europe. With more than 61 000 participants, the size of this study surpasses the combined 35 784 individuals described in a recent review of serosurveys.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. We found no differences in seroprevalence between females and males. Similar to what has been reported for other endemic coronaviruses, 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.The first wave of the study was done while Spain was under lockdown. Participants working in essential sectors did not show higher seroprevalence values than the general population, with the exception of health-care workers (specificity–sensitivity range 8·3–11·7%), who have been previously reported to have a greater exposure to SARS-CoV-2.
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.Our results confirm that close contact with people with COVID-19, and particularly those in the same household, increases viral transmission. Appropriate quarantine and separation from other household members can be particularly challenging and not realistic in urban areas and less affluent scenarios. While mass quarantine during the lockdown would reduce the number of potentially infective contacts, it would also increase the transmission of the virus in a confined space, as a recent simulation study has suggested. Serological surveys are the best tool to determine the spread of an infectious disease, particularly in the presence of asymptomatic cases or incomplete ascertainment of those with symptoms. 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. Seroprevalence was close to 90% after 14 days since a positive PCR test, which is consistent with a recent study concluding that SARS-CoV-2 IgG antibodies are detected in more than 90% of infected people 2 weeks after symptom onset, 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. 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.
One of the most practical conclusions from our survey is that, although the immunoassay had better performance features, our rapid point-of-care test yielded comparable epidemiological information while having a greater uptake, lower cost, and easier implementation. Thus, a high-performance point-of-care test could be a suitable option for large seroepidemiological studies. Additionally, as the two tests addressed different viral proteins, they might be providing complementary information. Differences in seroprevalence between our two tests among recently PCR-positive people could be compatible with a later appearance of IgG antibodies against the receptor binding domain of the S protein compared with those against the nucleoprotein.
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.35 In this sense, the combination of both tests provides a more conservative estimation of the real figures.
We focused on IgG antibodies, which last longer than IgM or IgA and are associated with viral neutralising activity. 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.
A key strength of our study is the random selection of households from the national municipal register (updated on Jan 1, 2020), which allowed us to contact a representative sample of the non-institutionalised Spanish population. However, this decision has its drawbacks: young adults have proven to be more difficult to find, probably due to their higher mobility, with many of them officially registered at their parents' home but living elsewhere. Also, some potential participants were staying at their second residences, leaving an empty house whose members could not be included. Moreover, household selection excludes care-home residents, who, according to recent estimations,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.
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