key: cord-0750964-11f5nsee
authors: Kaspersen, Kathrine Agergård; Greve, Thomas; Nielsen, Kent Jacob; Jespersen, Sanne; Mikkelsen, Susan; Vestergaard, Jesper Medom; Redder, Jacob Dvinge; Tolstrup, Martin; Thomsen, Marianne Kragh; Møller, Holger Jon; Østergaard, Lars; Kolstad, Henrik Albert; Erikstrup, Christian
title: Symptoms reported by SARS-CoV-2 seropositive and seronegative healthcare and administrative employees in Denmark from May to August 2020
date: 2021-06-11
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.06.017
sha: 01786cae419b30225c491117c3472181d70c27bc
doc_id: 750964
cord_uid: 11f5nsee

Background The distribution and nature of symptoms among SARS-CoV-2 infected individuals need to be clarified. Methods Between May and August 2020, 11,138 healthcare and administrative personnel from Central Denmark Region were tested for SARS-CoV-2 antibodies and subsequently completed a questionnaire. Symptom prevalence and overall duration for symptoms persisting for more than 30 days were calculated. Logistic regression models were used to estimate adjusted odds ratios (ORs) with 95% confidence intervals (CIs). Results In total, 447 (4%) of the participants were SARS-CoV-2-seropositive. Loss of sense of smell and taste was reported by 50% of seropositives compared with 3% of seronegatives. Additionally, seropositives more frequently reported fever, dyspnoea, muscle or joint ache, fatigue, cough, headache and sore throat, and they were more likely to report symptoms persisting for more than 30 days. In adjusted models, they had a higher risk of reporting symptoms with the strongest association observed for loss of sense of taste and smell (OR = 35.6; 95% CI: 28.6–44.3). Conclusion In this large study, SARS-CoV-2-seropositive participants reported COVID-19-associated symptoms more frequently than those who were seronegative, especially loss of sense of taste and smell. Overall, their symptoms were also more likely to persist for more than 30 days.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) is unprecedented in scale. In spite of the vigorous research in COVID-19, many questions about its symptom prevalence and long-term consequences remain unanswered [1] . During recent months, much research has been devoted to characterizing symptoms associated with COVID-19. However, their distribution and nature (infection morbidity rate) remain to be clarified. Studies on hospitalised patients report symptoms such as fever, cough, headache, muscle ache, nausea or vomiting, diarrhoea and shortness of breath [2, 3] . Some symptoms persist for weeks after discharge, especially fatigue, dyspnoea and anosmia [4] [5] [6] [7] . Non-hospitalised patients report similar symptoms [8] , and it is suggested that these symptoms may last for weeks or months [9, 10] .

The present study aimed to contribute to the growing knowledge of COVID-19 by exploring prevalences of COVID-19-associated symptoms obtained from self-reported questionnaires in a large cohort of 11,138 participants with mild and asymptomatic infections. We included healthcare and administrative employees from Central Denmark Region tested for antibodies against SARS-CoV-2 between May and August 2020. pandemic. The first known case of COVID-19 in Denmark was diagnosed February 27, 2020 [11] and the incidence peaked in March. The number of SARS-CoV-2-infected patients admitted to hospital peaked around April 1 st . This first wave was followed by low incidence for several months. A second wave affected Denmark from November 2020 through February 2021. During the second wave, the incidence of new infections peaked in December and admissions to hospital peaked at approximately 980 per day by the end of December [12] . Since the beginning of the pandemic, Denmark has used different strategies in controlling infection spread such as partial-lock downs, distancing measures, the use of protective equipment, etc. As of June 8, 2021, 287,325 known COVID-19 cases have been registered in addition to 15,448 admissions and 2,520 deaths with COVID-19 [13] .

All healthcare and administrative personnel at hospitals, prehospital services and specialist practitioners in Central Denmark Region were offered a test for SARS-CoV-2 antibodies. They were also invited to participate in this study by email and, if consent was given, to fill out a questionnaire.

Questionnaire data were collected from 15 May until 19 August 2020 and blood samples for SARS-CoV-2 antibody test were collected from 18 May until 19 June 2020.

The questionnaire addressed a wide range of items including sex, age, current smoking status, Body Mass Index (BMI), certain coexisting chronic diseases and a detailed list of COVID-19-related symptoms and their duration together. The participants were asked the following questions: "Have you had any of the following symptoms since March 1? More than one symptom is allowed". -Answer J o u r n a l P r e -p r o o f options: "fever", "sore throat", "cough", "dyspnoea", "headache", "muscle or joint ache", "fatigue", "loss of sense of taste and smell". "When did the symptoms begin?" -Answer option: a specific date.

"Do you still have symptoms?". -Answer options: "Yes", "no". "When did the symptoms stop?" -Answer option: a specific date. Hence, the symptoms were reported as a period prevalence, and the duration of each separate symptom was not available from this questionnaire.

The symptoms presented in this study have previously been associated with COVID-19, and we here report some of the symptoms most often mentioned by the study population.

In sensitivity analyses, we used the following questions regarding chronic diseases: "Do you suffer from (or receive medication in the treatment of) the following chronic diseases? More than one condition is allowed". -Answer options: "no", "asthma", "diabetes", "chronic lung diseases in addition to asthma (e.g., COPD, emphysema and other respiratory tract conditions)", "heart disease", "kidney disease", "weakened immune system due to medical treatment or chronic conditions such as complete or partial removal of the spleen, organ transplantation, HIV/AIDS, cancer, severe arthritis, etc.", "high blood pressure (hypertension)".

The questionnaire items used in this study are further described in the Supplementary Material.

A previous serosurvey has described the demographic characteristics of the participants and given a detailed description of blood sampling, real-time polymerase chain reaction (RT-PCR) testing, serological testing and seroprevalence distribution in the region for this cohort [14] .

After blood samples were collected, experienced staff tested undiluted EDTA plasma for IgG, IgM and IgA antibodies to the SARS-CoV-2 receptor-binding domain using a commercial SARS-CoV-2 total antibody enzyme-linked immunosorbent assay (ELISA, Wantai Biological Pharmacy Enterprise J o u r n a l P r e -p r o o f Co., Ltd., Beijing, China). The method was previously described in details [14] . The sensitivity of the assay is 96.7% and its specificity is 99.5% [15] .

A total of 25,950 employees were invited to participate, and 17,971 reported for antibody testing.

Among the 17,971 participants with a SARS-CoV-2 serological test, 11,138 agreed to participate in the survey and filled in the questionnaire. Participants with missing response to questionnaire, missing blood samples or poor blood sample quality were excluded. Analyses with adjustment for BMI and current smoking status were performed in 10,776 participants, as a total of 362 participants had missing responses to either BMI or smoking status. Thus, they were excluded from the analyses, see

From 29 February to 1 August 2020, a sub-group of the study population was tested for SARS-CoV-2 RNA using RT-PCR technique in case of relevant COVID-19 symptoms or risk of exposure to SARS-CoV-2 transmission (n = 3,690). Of these, a substantial part (n = 2,954) had been tested for SARS-CoV-2 RNA before being tested for SARS-CoV-2 antibodies.

The prevalence of each of the eight symptoms was reported and compared by SARS-CoV-2 serological status (positive or negative). Furthermore, the overall duration of symptoms persisting for more than 30 days was calculated in two ways: 1) number of participants reporting one or more symptoms persisting after 30 days among participants reporting the specific symptom (example given for seropositives -loss of sense of taste and smell: out of 218 seropositive participants reporting this specific symptom, 111 (51%) reported any symptom persisting for more than 30 days); 2) percentage of participants who reported both the specific symptom and one or more symptoms persisting after 30 days (example given for seropositives -loss of sense of taste and smell: 50% x 51% = 26%).

Prevalences excluding all SARS-CoV-2 RNA-positive participants were used in sensitivity analysis to assess the effect of biased recall of symptoms among participants already aware of their SARS-CoV-2 status.

In the calculation of overall duration of symptoms persisting for more than 30 days, participants were excluded if their symptoms started within 30 days of the completion of the questionnaire. This was done to avoid uncertainty about the duration of symptoms (246 participants were excluded from the main analyses and 243 participants from the sensitivity analyses, which also excluded all SARS-CoV-2 RNA-positive participants). Additionally, participants who reported start date after end date of symptoms were excluded (48 participants were excluded from the main analyses and 44 participants from the sensitivity analyses, which also excluded all SARS-CoV-2 RNA-positive participants). In total, 292 participants were excluded in the calculation of overall duration of symptoms persisting for more than 30 days in the main analyses, and the corresponding number of excluded participants in the analyses excluding all SARS-CoV-2 RNA-positive participants was 285 participants.

A multivariable logistic regression model was used to investigate the association between SARS-CoV-2 serological status and symptom reporting with adjustment for potential confounders. The following covariates, obtained from the questionnaire, were considered to adjust for potential confounding: sex (categorical: woman or man), age (categorical: 18 -39, 40 -59, ≥ 60), BMI (categorical: non-obese defined as a BMI below 30 kg/m 2 , obesity defined as a BMI exceeding or equal to 30 kg/m 2 ) and current smoking status (categorical: non-current smoker, current smoker).

Two confounder models were used: A basic model, by design adjusting for sex and age, and a fully adjusted model with further adjustment for BMI and current smoking status.

Furthermore, sensitivity analyses were performed for participants with or without self-reported chronic diseases based on the basic model. Participants reporting any of the above-mentioned chronic diseases were classified as having a chronic disease.

The prevalence of each of the eight symptoms by serological status is presented in Table 2a. SARS-CoV-2-seropositive participants were more likely than seronegatives to report all the symptoms (a p-value below 0.001 was observed for each of the symptoms). Notably, 50% of seropositives reported loss of sense of taste and smell compared with only 3% of seronegatives. Similarly, 53% of seropositives reported fever compared with 11% of seronegatives. Overall, 86% of seropositives reported any symptom compared with 55% of seronegatives. Seropositives who reported any of the specific symptoms were more likely than seronegatives also to have a duration of any symptom for more than 30 days (39% versus 22%). A total of 14% of seropositive participants reported no symptoms.

As described above, 3% (n = 293) reported loss of sense of taste and smell among the seronegatives.

Half of them were tested by RT-PCR and fewer than five seronegative participants were tested positive for SARS-CoV-2 RNA by RT-PCR. Thus, false negative serological test results could not be ruled out.

In a sensitivity analysis excluding 239 participants previously testing positive for SARS-CoV-2 RNA by RT-PCR, we found similar although attenuated effects of seropositivity (Table 2b) .

In models adjusted for sex and age, loss of sense of taste and smell was strongly associated with SARS-CoV-2 serological status (OR = 35.6; 95% CI: 28.6 -44.3) followed by fever, dyspnoea, muscle or joint ache, fatigue, cough, headache and sore throat (Figure 1 ). Further adjustment for BMI and current smoking status did not alter these results (confounder model 2). Results from confounder model 2 and The results from the sensitivity analyses with participants with or without a chronic disease are presented in Supplementary Material - Table 5 . The results were almost identical to the results from the main analyses. However, with slightly broader 95% CIs for participants with chronic disease.

To our knowledge, this study is one of the first large studies reporting COVID-19-associated symptoms among SARS-CoV-2-seropositive and seronegative participants. We included 11, 138 healthcare and administrative personnel from hospitals, prehospital services and specialist practitioners from Central Denmark Region who were screened for SARS-CoV-2 antibodies and subsequently completed a questionnaire. The study was conducted between May and August 2020 after the first wave and before the second wave of the COVID-19 pandemic in Denmark.

We found that 50% of seropositive participants reported loss of sense of smell and taste with only very few seronegative participants reporting similar symptoms. More than 50% of seropositive participants reported fever.

The sensitivity of the assay used in this study was 96.7% and the assay with the highest sensitivity among 16 validated SARS-CoV-2 immunoassays in a Danish national validation study [15] . In a previous report, we were further able to verify this high sensitivity: 98% of employees previously tested positive for viral RNA by RT-PCR had a positive test for SARS-CoV-2 antibodies using this test assay [14] . The present study is thus unique as it also includes participants with mild and asymptomatic infections. This enables us to estimate the absolute percentage of individuals with specific symptoms. Among the seropositives, 14% reported none of the explored symptoms while this was the case for 45% of the seronegative participants. The true percentage of asymptomatic SARS-

CoV-2 infection cases may be even larger, and asymptomatic carriers may contribute to infection spread.

In models adjusting for sex and age, seropositives had a distinctly higher risk of reporting symptoms with ORs above 5 for each of the symptom categories, loss of sense of taste and smell, fever, dyspnoea, muscle or joint ache and fatigue. These findings changed inconspicuously after further adjustment for BMI and current smoking status. Even in sensitivity analyses, stratified for self-reported chronic disease, the findings were consistent indicating that our findings were not affected by these potential confounders. No marked differences between women and men were observed.

The most prominent symptom associated with seropositive status in our study was loss of sense of taste and smell followed by fever. These findings complement those of a recently published study among 2,149 Swedish healthcare workers. However, no measure of symptom duration was available from that study [16] . Other studies similarly showed that alterations in smell or taste were frequently reported by mildly symptomatic patients with SARS-CoV-2 infection and were often the first apparent symptom [17, 18] . We were, however, able to confirm that these effects persisted even after exclusion of SARS-CoV-2 RNA-positive participants. This sensitivity analysis was performed to determine any potential recall bias: Participants already aware of having had SARS-CoV-2 infection may report COVID-19-associated symptoms differently from participants who are not aware of having had SARS-CoV-2 infection simply because of the increased focus on COVID-19 symptoms during recent months.

Along with this, only 0.5% of the participants responded to the questionnaire after the serological test result was sent to them.

With respect to persisting symptoms, we found that seropositives were more likely than seronegatives to report symptoms lasting more than 30 days. In comparison, a study found that among COVID-19recovered patients, 87.4% reported persistence of at least one symptom, particularly fatigue and dyspnoea [4] . Another study showed that 43.4% of COVID-19 cases had symptoms lasting longer than

A major strength of this study is our large study population consisting of 11,138 healthcare and administrative personnel screened for SARS-CoV-2 antibodies. It is a limitation, that only employees with a current labour market affiliation were included. Should employees have been incapacitated due to severe COVID-19 they are not part of this survey.

The questionnaire, as previously explained, was filled before the result of the antibody test was available among the vast majority of participants. It is a strength of the study that we, furthermore, had the possibility to perform sensitivity analyses excluding participants with a positive RT-PCR for viral RNA. This analysis seeks to minimize recall bias by assessing effects among participant with no prior positive tests.

Most of the explored symptoms are not specific to COVID-19 and could be explained by other conditions and common viruses. Findings among both SARS-CoV-2-seropositive and seronegative participants may therefore be affected by other common infections during the study period. For example, it is well-know, that also other virus infections can cause the same symptoms and especially parainfluenza virus can cause alterations in sense of smell [20] .

It is possible, that some participants developed infection and antibodies after filling out the questionnaire underestimating the symptom prevalence. However, 68% of the participants completed the questionnaire before or at the same date as the antibody test was performed. and 99% of the participants were tested within 16 days of responding. When allowing for at least a week from infection to antibody development and that the survey was performed during low incidence of new SARS-CoV-2 infections the risk of this bias seems low [21] .

We had information on age, BMI, current smoking status and certain self-reported chronic diseases, which allowed us to take into account these potential confounders. However, the interference of other comorbidities and calculations of comorbidities on a weighted scale, for example by using the J o u r n a l P r e -p r o o f symptoms were self-reported and thus prone to recall bias.

A major limitation is the lack of data on duration for each specific symptom. Still, we observed that seropositives were more likely than seronegatives to report symptoms persisting for more than 30 days.

In the health care system in Central Denmark Region, 80% of the employees are women. In this study, women comprised 88% of the participants. Thus, only a part of the men we could expect to include in this study actually did participate.

In analyses stratified by sex, we saw no overt differences between women and men; but with such a low percentage of men in the study, the statistical power to detect differences between sexes is low.

A substantial share (38%) of the study population tested for SARS-CoV-2 antibodies did not complete the questionnaire, contributing to potential selection bias. Symptoms among participants in the serosurvey may have affected their willingness to participate. Several possible biases may exist because symptomatic infections may have affected the chance of participation. However, in the serosurvey with descriptive data on healthcare and administrative personnel from hospitals, prehospital services and specialist practitioners from Central Denmark Region including our cohort, the seroprevalence was 3.7% among the 17,917 participants [14] . Compared with the seroprevalence of 4.0% (95% CI: 3.5-4.4%) among 11,138 participants included in this study, the estimated seroprevalence is 3.3% (95% CI: 2.9-3.7%) for the 38% of the participants not participating in our study.

In this large study of healthcare and administrative employees, loss of sense of smell and taste was reported among 50% of SARS-CoV-2-seropositive participants compared with 3% among seronegative participants. In addition, fever, dyspnoea, muscle or joint ache, fatigue, cough, headache and sore throat were more frequently reported by seropositive participants. The duration of any J o u r n a l P r e -p r o o f Data presented as numbers with percentages.

a Number of participants reporting one or more symptoms persisting for more than 30 days among participants reporting the specific symptom.

b Percentage of participants who both report the specific symptom and report one or more symptoms persisting after 30 days.

c Numbers in parentheses represent participants reporting the symptom (corresponding to the number of "Reported symptoms") but excluding participants whose symptoms started within 30 days of the completion of the questionnaire (n = 243) in addition to participants with discrepancy between symptom start date and symptom end date (n = 44). In total, 285 participants were excluded.

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Age and BMI are further presented as medians with interquartile ranges (IQR)

To compare groups, t-tests were used for normally distributed data and Mann-Whitney U tests were used for non-normally distributed data. Chi-squared tests were used to compare categorical values

The authors thank all staff involved in performing this study. Furthermore, the authors thank all healthcare and administrative personnel at hospitals, prehospital services and specialist practitioners in the Central Denmark Region participating in this study.J o u r n a l P r e -p r o o f