key: cord-0726681-5sudowhg
authors: van den Hurk, Katja; Merz, Eva-Maria; Prinsze, Femmeke J.; Spekman, Marloes L.C.; Quee, Franke A.; Ramondt, Steven; Slot, Ed; Vrielink, Hans; Huis in ’t Veld, Elisabeth M.J.; Zaaijer, Hans L.; Hogema, Boris M.
title: Low awareness of past SARS-CoV-2 infection in healthy plasma donors
date: 2021-03-01
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2021.100222
sha: c50fcc9d01a00d1f0e5bff2ad17aa6cda43d74ee
doc_id: 726681
cord_uid: 5sudowhg

Awareness of infection with SARS-CoV-2 is crucial for the effectiveness of COVID-19 control measures. Here, we investigate awareness of infection and symptoms in relation to antibodies against SARS-CoV-2 in healthy plasma donors. We ask individuals donating plasma across the Netherlands between May 11th and 18th 2020 to report COVID-19 related symptoms and we test for antibodies indicative of a past infection with SARS-CoV-2. Among 3,676 with antibody and questionnaire data 239 (6.5%) are positive for SARS-CoV-2 antibodies. Of those, 48% suspect no COVID-19 despite the majority reporting symptoms. 11% of seropositive individuals report no, and 27% very mild symptoms at any time during the first peak of the epidemic. Anosmia/ageusia and fever are most strongly associated with seropositivity. Almost half of seropositive individuals do not suspect SARS-CoV-2 infection. Improved recognition of COVID-19 symptoms, in particular anosmia/ageusia and fever, is needed to reduce widespread SARS-CoV-2 transmission.

Due to the current coronavirus disease pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), governments worldwide are struggling to find an appropriate balance between virus control measures and their societal and economic consequences. 1 Physical distancing and (partial) closures of offices, nursing homes, restaurants, schools and shops have played -and still play -an important role in combating the spread of SARS-CoV-2. An impending economic crisis and the huge societal burden call for informed easing of these measures.

Currently, limited knowledge exists regarding the extent to which SARS-CoV-2 infections may remain undetected, while pre-and asymptomatic individuals are thought to contribute significantly to the spread of SARS-CoV-2. 2,3 A wide clinical spectrum of SARS-CoV-2 infections has been described, ranging from mild flu-like symptoms to severe viral pneumonia with respiratory failure, and death. 4, 5 Due to the limited availability of tests and infrastructure, more severe COVID-19 cases are likely overrepresented in the majority of studies conducted thus far. Many cases may remain undetected in the event of asymptomatic infection, mild infection with isolated symptoms such as the loss of taste and/or smell (anosmia/ageusia), or symptomatic infections that are attributed to other causes. 3, 6, 7 Post-lockdown measures often rely on individuals, in particular those who have been in contact with a confirmed COVID-19 case, to self-isolate and get tested in the event of COVID-19-related symptoms. These measures are dependent on individuals' recognition of symptoms, yet it is unknown whether or not infected individuals are able to identify themselves as such. Hence, we studied the association between COVID-19 suspicion and SARS-CoV-2 antibody status, as well as that between self-reported symptoms and antibody status in healthy adults.

Of the 8,275 donors who underwent plasmapheresis between the 11 th and 18 th of May 2020, we tested 7,150 for SARS-CoV-2 antibodies, of whom 419 (5.9%) tested positive. OD/CO ratio in seropositive individuals ranged from 1.01 to 20.86. We invited 7,721 individuals to participate in the online questionnaire, of whom 4,275 (55.4%) participated. Antibody and questionnaire data were complete for 3,676 individuals, including 239 (6.5%) who tested positive for SARS-CoV-2 antibodies. Seropositive individuals were generally younger and more likely to live in the Southern region of the Netherlands than seronegative individuals (Table 1) . Forty-eight percent of the seropositive individuals, and 87% of those seronegative, did not suspect they had had COVID-19. About 11% of the seropositive individuals reported no symptoms at all, 73% reported symptoms indicative of COVID-19. An additional 27% of seropositive individuals reported only very mild symptoms, generally sneezing (69%), coryza (55%), and/or fatigue (40%). Only one individual positive for SARS-CoV-2 antibodies was admitted to a hospital, but this was because of gastrointestinal complaints. The median date of symptom onset in seropositive individuals was March 15, 2020. Symptom onset was between March 6 and 28 in 50% of the seropositive individuals.

Apart from diarrhoea, vomiting, rash, and an altered mental state (confusion), all symptoms were significantly more frequently reported by seropositive versus -negative individuals ( Figure 1 ). Anosmia/ageusia was the symptom most strongly associated with the presence of SARS-CoV-2 antibodies; the odds ratio of 12.7 was significantly higher than for any other COVID-19-related symptom ( Table 2) . Despite this strong association, it was not the most prevalent symptom among seropositive individuals. Other symptoms, such as fever, coryza, fatigue, cough, headache, myalgia, and sneezing were similarly or even more prevalent among these individuals. However, these symptoms appeared less indicative of a SARS-CoV-2 infection.

In seropositive individuals, the presence of symptoms was also significantly associated with high levels of SARS-CoV-2 antibodies, except for the symptoms sneezing and nausea ( Table   2 ). Dyspnoea and muscle weakness were most strongly associated with high levels of SARS-CoV-2 antibodies. Individuals who suspected having had a SARS-CoV-2 infection were significantly more likely to be SARS-CoV-2 antibody positive and, among those antibody-positive individuals, to have high levels of antibodies compared to individuals who did not suspect having had COVID-19. More severe symptoms versus being asymptomatic was also associated with antibody status and levels, as was consultation of a physician because of those symptoms.

Of all individuals who tested positive for SARS-CoV-2 antibodies, those who did not suspect having had the infection reported no or very mild symptoms in 19 and 41% of all cases, compared to only 3 and 14%, respectively, of those who did (Table 3) . Physician contact and hospital admittance did not differ between antibody-positive individuals who did or did not suspect having had COVID-19. Individuals who did not suspect having had COVID-19 attributed their symptoms to unrelated circumstances, such as allergies, in 39% of the cases, or temporary illness such as the flu, unknown reasons or chronic complaints in 20, 18 and 4% of the cases, respectively. Among the 45 individuals who did not suspect COVID-19 in spite of mild to severe symptoms, 5 reported a negative PCR test (tests were done 3 to 52 days after symptom onset). Nine individuals had non-specific symptoms (e.g. coryza, sneezing). Another 31 individuals had symptoms indicative of COVID-19 according to the WHO Case Definition, but attributed those to unrelated circumstances (e.g. hay fever or trauma, n=17), temporary illness (e.g. flu, n=8), chronic complaints (n=3) or unknown reasons (n=3). Of all individuals who tested negative for SARS-CoV-2 antibodies, those suspecting COVID-19 were significantly more likely to report more severe symptoms (76 versus 27%), or symptoms indicative of COVID-19 according to the WHO Case Definition (84 versus 33%).

We explored associations between COVID-19 suspicion and SARS-CoV-2 antibody status, as well as between symptoms and antibody status in healthy adults. Of those with reactive test results, 48% did not suspect having been infected with SARS-CoV-2. Eleven percent reported a complete absence of symptoms and 27% only very mild symptoms during the national peak of the epidemic. COVID-19-related symptoms -particularly anosmia/ageusia and fever -were significantly associated with antibody status, independent of age and gender.

The lack of COVID-19 suspicion in almost half of the subgroup that tested positive for SARS-CoV-2 antibodies may have an impact on individual adherence to governmental measures and on the decision to request a PCR test, as health behavior largely depends on beliefs regarding one's own and others' perceived susceptibility to and severity of disease. 13 A behavioral study by the National Institute for Public Health and the Environment showed that 80% of the people who reported symptoms did not stay inside their homes and 40% even went to work. 14 Also, while 68% of the study participants indicated that they will get tested if they develop symptoms, this percentage drops to 28% among individuals once they displayed symptoms. The main reason for this was that individuals attributed their symptoms to hay fever or a common cold, of which the latter may occur even more frequently during the flu season.

Assuming that the vast majority of SARS-CoV-2 infections induce an antibody response, 38% of the infected individuals reported having no (11%) or only very mild symptoms (27%). 15 An important advantage of our retrospective study design in comparison to previous studies reporting 40-45% asymptomatic infections is the unlikelihood of falsely identifying pre-symptomatic cases as asymptomatic. 3 In addition, our thorough assessment of symptoms over an extended period is likely to have captured milder symptoms that may have been underreported in previous studies. Indeed, previous studies have been mainly J o u r n a l P r e -p r o o f cross-sectional or with incomplete follow-up and with register-based symptoms rather than systematic assessments. 16, 17 Almost 13% of the individuals who tested negative for SARS-CoV-2 did suspect a SARS-CoV-2 infection, a large majority (84%) because of symptoms indicative of COVID-19. This finding further emphasizes the non-specificity of COVID-19 symptoms, although it cannot be excluded that some of these individuals had had COVID-19 without a detectable antibody response.

Particularly those individuals who reported no or very mild symptoms only were relatively unlikely to suspect having been infected with SARS-CoV-2. On the other hand, our study shows that in comparison to being asymptomatic, even very mild symptoms, and mild to severe symptoms in particular, were associated with SARS-CoV-2 infection, as well as with high levels of SARS-CoV-2 antibodies. Our finding that anosmia/ageusia is the symptom most strongly associated with COVID-19 is in line with results from the COVID Symptom Study, which showed this symptom to be the strongest predictor of PCR-confirmed SARS-CoV-2 infection. 18 Improved awareness and recognition of COVID-19 symptoms, in particular of the loss of smell and taste, may therefore help to reduce the proportion of undetected COVID-19 cases.

Strengths of this study include the large study sample, the superior performance of the Wantai SARS-CoV-2 total antibody ELISA over other antibody tests, and the thorough questionnaire-based assessment of the presence and severity of symptoms. 19 As governments slowly ease virus control measures, it becomes vital to identify and isolate infected individuals to prevent new SARS-CoV-2 outbreaks. 20, 21 The presence of anosmia/ageusia, especially, should trigger PCR testing. 18 In addition, our study confirms the existence of asymptomatic SARS-CoV-2 infections and adds that even symptomatic individuals did not suspect a SARS-CoV-2 infection. Despite the limitations of studies thus far, sufficient evidence of asymptomatic and pre-symptomatic transmission of SARS-CoV-2 exists. 17, 22 Efforts to identify cases that rely on symptoms may therefore be insufficient, which emphasizes the importance of thorough contact tracing. 23 J o u r n a l P r e -p r o o f

In conclusion, almost half of the individuals who tested positive for antibodies to SARS-CoV-2 in a high-performance immunoassay did not suspect having had an infection. This proportion may be lowered with better awareness and recognition of COVID-19 symptoms, in particular the loss of smell and taste. However, 38% of those infected reported no or only very mild symptoms. Tracing of asymptomatic contacts is therefore crucial to reduce widespread SARS-CoV-2 transmission.

Study participants were required to be in good health in order to qualify for plasma donation. Age and gender-adjusted logistic regression models indicate higher prevalence in 239 antibody positive versus 3437 -negative individuals for all symptoms (p<0.001, Table 2 ), except where indicated as not significant (NS). Samples that tested reactive for antibodies, including samples with weak reactivity (OD/CO ratio ≥0.5), were re-tested and considered positive if the re-test was reactive (OD/CO ratio >1.0). Tables   Table 1 J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f

Further information and requests for resources should be directed to and will be fulfilled by the Lead Contact, Katja van den Hurk (k.vandenhurk@sanquin.nl).

This study did not generate new unique reagents.

The dataset generated and analyzed in the context of this study is made available via the Open Science Framework (https://osf.io/xds75/). The computer code used to generate results that are reported in this paper are available from the Lead Contact upon request. Table 1 . To reduce any risk of social desirability bias, we have assured individuals before taking part in the survey that their data will be treated confidentially and that all data will be coded and never traced back to the individual.

Plasma samples were tested for the presence and levels of antibodies against SARS-CoV-2, using a SARS-CoV-2 total antibody ELISA (Wantai Biological Pharmacy Enterprise Co., Beijing, China) following the manufacturer's instructions. Samples that tested reactive, including samples with weak reactivity (OD/CO ratio ≥0.5), were re-tested and considered positive if the re-test was reactive (OD/CO ratio >1.0). We previously determined the sensitivity and specificity of the Wantai test to be 98.7% and 99.6%, respectively. 10 Questionnaire (in Dutch)'). 11 Participants indicated the extent to which they suffered from these symptoms in the period between one week before the first confirmed case nationally (February 21 st , 2020) and their donation date on a scale from 0 (not at all) to 5 (severely). A symptom was considered present if the score was 1 (very mild) or higher. Symptom severity was defined as asymptomatic (score 0), only very mild symptoms (score 1) or mild to severe symptoms (score for at least one symptom 2 or higher). In accordance with the World Health

Organization's COVID-19 Case Definition, clinically suspected COVID-19 was defined based on clinical criteria. This implies that individuals who reported either anosmia/ageusia, or fever and cough, or three or more of the following symptoms: fever, cough, general weakness/fatigue, headache, myalgia, sore throat, coryza, dyspnoea, anorexia/nausea/vomiting, diarrhoea, altered mental status, were considered clinically suspected COVID-19 cases. 12 Participants who reported symptoms were also asked whether or not they consulted a physician or were admitted to a hospital and what they thought had caused their symptoms:

temporary illness (e.g. flu, COVID-19), chronic complaints, other circumstances (e.g. allergies, trauma) or unknown.

Demographic background data on age, gender, and region were obtained from the blood bank information system eProgesa (MAK systems, Paris, France).

Descriptive information for continuous variables was calculated as mean and standard deviations, or median and interquartile range if skewed. We performed two-sided t-tests for continuous variables and chi 2 tests for proportions to assess differences between subgroups.

Associations between symptoms and their severity with antibody status and levels were estimated using logistic regression analyses. Statistical analyses were performed using SPSS, Version 23 (IBM, Armonk, U.S.A.).

Online questionnaire (in Dutch).

J o u r n a l P r e -p r o o f

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Self-reported olfactory loss associates with outpatient clinical course in COVID-19

Probability of symptoms and critical disease after SARS-CoV-2 infection. arXiv (2020). [online preprint] 8 National Institute for Public Health and the Environment

Guide to the preparation, use and quality assurance of blood components. Recommendation no. R(95)15

Low SARS-CoV-2 seroprevalence in blood donors in the early COVID-19 epidemic in the Netherlands

World Health Organization. WHO COVID-19: Case Definitions. WHO reference number

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Asymptomatic Transmission, the Achilles' Heel of Current Strategies to Control Covid-19

CoV-2 antibody testing in healthy blood donors reveals a lack of awareness of past infection • 48 % of SARS-CoV-2 seropositive individuals do not suspect infection

• 11% of SARS-CoV-2 seropositive individuals report no symptoms and 27% only very mild symptoms

• Anosmia/ageusia is 12.7 times more common in seropositive individuals

investigate awareness of SARS-CoV-2 infection and symptoms in relation to antibody status in healthy plasma donors. Forty-eight percent of seropositive individuals do not suspect SARS-CoV-2 infection, 11% are asymptomatic. Improved recognition of COVID-19 symptoms, in particular anosmia/ageusia and fever, is needed to reduce SARS-CoV-2 transmission. Summary Awareness of infection with SARS-CoV-2 is crucial for the effectiveness of COVID-19 control measures. Here, we investigate awareness of infection and symptoms in relation

We ask individuals donating plasma across the Netherlands between May 11 th and 18 th 2020 to report COVID-19 related symptoms and we test for antibodies indicative of a past infection with SARS-CoV-2. Among 3,676 with antibody and questionnaire data 239 (6.5%) are positive for SARS-CoV-2 antibodies

The study has been funded by Sanquin Blood Supply Foundation and the Dutch Ministry of Health, Welfare and Sport. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors declare no competing interests.