Outlining the Prior Infection with SARS-CoV-2 study (PICOV) - preliminary ﬁndings on symptoms in nursing home residents and staﬀ

Background: COVID-19 has presented itself as one of the most important health concerns of 2020. The geriatric population is arguably hit the hardest by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The ”Prior Infection with SARS-COV-2” (PICOV) study investigates both residents and staﬀ members from nursing homes. The primary aim of the study is to compare the time to occurrence of an inﬂuenza-like illness (ILI) or acute respiratory infection (ARI) between participants with a conﬁrmed past SARS-CoV-2 infection and those without an infection. This paper details the study design, sampling scheme, biological measurements, and population characteristics at baseline. Methods: In 26 Belgian nursing homes, all eligible residents and staﬀ members with or without a past SARS-CoV-2 infection (ratio 40/60) were invited to participate. Consent was obtained from 1,375 participants and 1,226 completed the baseline questionnaire. Prevalence of symptoms during a prior SARS-CoV-2 infection was compared between residents and staﬀ members with χ 2 statistics. Results: Nursing home residents (both with and without a prior SARS-CoV-2 infection) systematically reported fewer symptoms than staﬀ members. Moreover, results from prior nasopharyngeal RT-qPCR and baseline serology show that antibody development after a SARS-CoV-2 infection diﬀers between residents and staﬀ members. Conclusions: We can postulate that disease development and symptoms is diﬀerent between a geriatric and younger population. Therefore, the occurrence and severity of a future ILI and/or ARI might vary from resident to staﬀ.


Background
At the end of 2019, a novel coronavirus causing acute respiratory disease  emerged in the Wuhan region of China and has since led to a worldwide pandemic. The causative agent was named 'severe acute respiratory syndrome coronavirus 2' (SARS-CoV-2) and is closely related to SARS-CoV, which led to an epidemic between 2002 and 2004 [1]. It is estimated that around 40-45% of all SARS-CoV-2 infections remain asymptomatic [2], for which an dependency on age was observed [3]. Those who do develop COVID-19 present variable clinical out-comes, ranging from mild disease with typical symptoms including fever and cough to severe respiratory illness and death [4,5,6,7].
COVID-19 related mortality is strongly age-dependent, with the highest incidence of deaths reported in the geriatric population [6,8,9]. Comorbidities related to ageing as well as immunosenescence, i.e. decreased immunological competence due to biological ageing, lie at the basis of this increased risk of death with advanced age [10]. In Belgium, around 10% of the population aged 65 and older lives in nursing homes where they receive formal long-term care [11], and because of this high concentration of susceptible people in such facilities, more than 60% of all COVID-19 related deaths between March and June 2020 occurred in nursing home residents [12].
In addition, multiple studies have shown significant co-infection rates of SARS-CoV-2 infected patients with viruses causing influenza-like illnesses (ILI) or acute respiratory tract infections (ARI) such as seasonal mild corona viruses, influenza A/B virus, parainfluenzavirus, rhinovirus, bocavirus, human metapneumovirus, and adenovirus [13,14,15,16]. As is the case for COVID-19, geriatric people are more at risk for severe clinical forms of these infections. While there are indications that coinfection with e.g. influenza A might increase SARS-CoV-2 viral loads and thereby someone's degree of contagiousness [17], in general little is known about the impact of one infection (previous or current) on the other in terms of susceptibility, immune response and clinical course.
The primary objective of this multi-centre prospective cohort study is to assess whether a confirmed prior infection with SARS-CoV-2 (PICOV) affects the susceptibility to and severity of an ILI and ARI. The aim of the current paper is to describe the PICOV study design, sampling scheme, biological measurements as well as baseline population characteristics.

Study design
The PICOV-study is a multi-centre prospective cohort study conducted in 26 nursing homes located in the Belgian regions of Flanders, Wallonia, and Brussels. Both residents as well as staff were eligible to participate. Inclusion of an equal number of participants with and without a previous SARS-CoV-2 infection is targeted.

Inclusion criteria
• Aged 18 years or older.
• Having a Belgian National Register Number.
• Being insured by one of the Belgian sickness funds.
• Having the cognitive ability to give consent to participate.

Exclusion criteria
• The inability to complete questionnaires in Dutch or French.
• Life-expectancy shorter than the duration of the study.
• Inaccessible veins for simple periphery venipuncture.
• Individuals who will not work or reside at the nursing home for the full duration of the study. • Individuals who had a previous diagnosis of dementia, having a mini-mental state examination (MMSE) of 18/30 or less.

Objectives of the PICOV study
The primary objective of the PICOV study is to compare the susceptibility to and the severity of an ILI and ARI in subjects who have had a confirmed prior infection with SARS-CoV-2 compared to those who have not experienced a SARS-CoV-2 infection. The aim of the present manuscript is to describe the study design, sampling scheme, biological measurements, and population characteristics at baseline. Additional study objectives include: • Determining which viruses are responsible for the ILI or ARI incidences throughout the study. • Evaluating whether COVID-19 convalescent participants are protected against reinfection with SARS-CoV-2 and identifying serological markers that are associated with protection.
• Assessing the influence of a previous SARS-CoV-2 infection on the immunological response to influenza vaccination.

Recruitment
Eligible staff members and residents with a documented previous SARS-CoV-2 infection were invited to participate. A serological screening with an antibody rapid diagnostic test (RDT) was performed for those who never received a positive RT-qPCR or serological test. Antibody RDTs included the CORIS COVID-19 Ab Rapid Test (CORIS BioConcept, Gembloux, Belgium), COVID-PRESTO ® COVID-19 IgG/IgM Rapid Test (AAZ-LMB, Boulogne-Billancourt, France), and the quickZen COVID-19 IgG/IgM Rapid Test (Zentech, Liège, Belgium). Those with a positive RDT result were also invited to participate. Next, approximately 20% more staff and residents without a documented SARS-CoV-2 infection or positive antibody RDT result were invited and enrolled in the study in order to compensate for asymptomatic infections and false-negative antibody RDT results to reach our target of 50% with and 50% without a past SARS-CoV-2 infection. In nursing homes which reported that over 50% of their residents and staff ever had a positive SARS-CoV-2 RT-qPCR test, no prior RDT screening was done and all eligible participants were immediately invited to participate. Dates and results of previous lung CT-scans, RT-qPCR tests and/or antibody tests were collected by the study nurses.

Sample collection and biological measurements
Biological samples, including nasopharyngeal swabs, saliva, and blood, were collected from all participants at baseline and at the end of the study. Figure 2 summarises which samples are collected at each study visit. Participants who were vaccinated against seasonal flu had an extra sampling visit, two weeks after vaccination. The flu vaccines which were administered during the study period included Vaxigrip Tetra (Sanofi S.A., Gentilly, France) and Influvac Tetra (Abbot Biologicals B.V., Olst, The Netherlands). In addition, study participants presenting with symptoms of an ILI or ARI had two extra sampling visits: one within five days of symptom onset and another two to three weeks after symptom onset during the attenuation of their illness.
Nasopharyngeal swabs (stored in universal transfer medium -UTM ® : Viral Transport [Copan, California, USA]) were collected to detect an ongoing SARS-CoV-2 infection by RT-qPCR. Serum (8 mL CAT Serum Separator Clot Activator Vacuette ® Tubes, Greiner Bio-One, Kremsmünster, Austria) was collected for serological measurements. Heparinised blood (x 10 mL plastic BD Vacutainer Lithium Heparine tubes (BD, New Jersey, USA) was collected for plasma and peripheral blood mono-nuclear cell (PBMC) isolation for cellular immunity studies. Oral fluid (Oracol Saliva System, Malvern Medical Developments Ltd, Worcester, UK) was collected to study mucosal immunity.

Questionnaires
Participants completed questionnaires at baseline, at the end of the study and in the case an ILI or ARI occurs. Staff members received a personal link to fill out the questionnaires themselves on-line, while residents were assisted by the study nurses.

Baseline
The questionnaire at baseline provides detailed basic information about participant age, gender, self-reported weight and height, ethnicity, smoking status, and physical activity. Medication use, prior SARS-CoV-2-test results (i.e. RT-qPCR, lung CTscan, and/or serology), vaccination status (i.e. pneumococcal vaccination in the past five years and influenza vaccination in the past year) are also recorded. In addition, the profession of staff members as well as the Katz-scale and MMSE-score of residents were also recorded.
Finally, participants were required to answer questions regarding any previous COVID-19 episode or influenza-like symptoms. These questions recorded the symptoms, their duration, the impact of COVID-19 on their daily activities and the severity of the illness as approximated by hospitalisation and/or ventilation requirements.
Standardised questionnaires were also provided and included a standardised measure for the health status: EQ-5D five-level (EQ-5D-5L) by EuroQol [18]. For residents, frailty was assessed via the 9-point Clinical Frailty Scale [19].

End of study
The questionnaire at the end of the study collects, besides current weight, smoking status and physical activity of the participants, information on COVID-19 episodes or influenza-like symptoms that occurred during the study period. Similar to the baseline questionnaire, (duration of) symptoms, impact of COVID-19 on daily activities and severity of illness are recorded.
The standardised questionnaires are collected once more: EQ-5D-5L for both staff and residents, and the 9-point Clinical Frailty Scale for only the residents.

Biological measurements at baseline
Nasopharyngeal swabs tested for the presence of SARS-CoV-2 RNA with an RT-qPCR targeting the E gene, according to the protocol by Corman et al. [20]. The Ct cut-off for positivity was 40.
The Wantai SARS-CoV-2 Ab ELISA (cat n°WS-1096; Beijing Wantai Biological Pharmacy Enterprise Co. Ltd., China) detects anti-receptor binding domain (RBD) IgG, IgA and IgM simultaneously in serum. In house and reported validations show a test specificity of 99.6% and a sensitivity of 100% at 14 days post-clinical illness onset [21,22,23].

Statistical analysis
Database management and data analysis were performed with the R software (version 4.0.3.). Descriptive statistics are used to describe the baseline characteristics. Mean ± standard deviation (±SD) is given for continuous variables and the proportion (%) for categorical variables. Normality of data distributions was tested with the Shapiro-Wilk statistics.
We assessed the proportions of categorical variables (χ 2 -statistics). The Mann-Whitney test was performed for the analysis on number of symptoms, as the data was not normally distributed.

Recruitment and sample size
Recruitment of participants started on September 24 th , 2020, and ended on December 8 th , 2020. A total of 1,375 participants initially provided informed consent to participate ( Figure 1). 116 individuals (8.4%) withdrew from the study resulting in a final sample size of 1,226 participants. Baseline sampling was completed on December 8 8 , 2020) and sampling after flu vaccination was completed on January 25 th , 2021).

Residents
The 374 residents had an average age of 81.9 years (±10.7) and a mean BMI of 25.5 kg/m 2 (±5.3). More than two-thirds was female (64.4%), and 99.2% was of European descent. Most residents never smoked (72.2%).

Staff
Staff members had an average age of 44.2 years (±11.5) and mean BMI of 26.1 kg/m 2 (±4.8). The majority was female (85.3%) and 747 were of European descent (87.7%). 619 (72.7%) staff members reported to have never smoked. Nurses (23.2%) and nursing assistants (29.7%) were the most included professions (Table 3). In total, 52 (6.1%) staff members worked in specialised COVID departments within their respective nursing homes. 27 of them indicated to have worked solely in these (temporary) departments.

SARS-CoV-2 history at baseline
Previous nasopharyngeal RT-qPCR and serology data indicated that 445 participants reported to have had a positive SARS-CoV-2 RT-qPCR or serology test between March and August 2020, while 781 reported no or only negative RT-qPCR or serology tests (Table 2). Taking serology at baseline into account, 586 participants (47.8%) had neither a positive SARS-CoV-2 RT-qPCR nor a positive anti-RBD IgG antibody test and are therefore categorised as 'no past SARS-CoV-2 infection'. The remaining 640 participants (52.2%) are categorised as 'past SARS-CoV-2 infection'. Of the latter group, 61 (9.5%) had a positive RT-qPCR but no detectable antibodies at baseline, 182 (28.4%) never had a positive RT-qPCR but did have detectable antibodies at baseline, and 397 (62.0%) had a previous positive RT-qPCR result and detectable antibodies at baseline.

Symptoms from March to August 2020
Participants with a past SARS-CoV-2 infection reported significantly more symptoms than those without an infection (p < 0.0001), at 5.5 (±3.9) and 3.0 (±2.3) symptoms, respectively ( Figure 3). This effect was the strongest in the staff members, as those with a past infection had 6.1 (±4.0) symptoms compared to 3.1 (±2.2) symptoms for staff members without an infection (p < 0.0001). In residents, a similar effect was observed as the number of symptoms during a past SARS-CoV-2 infection was higher compared to those without an infection (3.1 vs 1.9 symptoms, p = 0.0155).

Persisting symptoms at baseline sampling
Participants with a past SARS-CoV-2 infection were asked whether they experienced any more symptoms at baseline sampling (approximately one to ten months after infection;

Discussion
The PICOV study is a prospective multi-centre study designed to investigate the effects of a prior SARS-CoV-2 infection on the occurrence and severity of ILI and ARI among residents and staff members of nursing homes. At the moment of publication, enrolment, baseline sampling, and follow-up sampling after vaccination have been completed. Comparing the symptoms between nursing home residents and staff members showed some interesting differences. On the one hand, residents have a higher proportion of asymptomatic cases (i.e. negative prior RT-qPCR results and detectable Ab at baseline) than staff members. On the other hand, staff members had a higher percentage of prior positive RT-qPCR swabs. Moreover, staff members with a past SARS-CoV-2 infection systematically report more symptoms than either residents with a past infection and staff members without a past infection. In contrast, residents with a past SARS-CoV-2 infection reported slightly more symptoms than residents whitout a past infection, albeit statistically not significant.
Fatigue and fever were the most prevalent self-reported symptoms during a 'past SARS-CoV-2 infection', accounting for over a third of nursing home residents. A recent systematic review by Neumann-Podczaska and colleagues described symptom data of 1,285 older people (aged 60 years and over) from 20 studies [24]. The authors reported that fever and cough were the most common symptoms at 83.6% and 62.7%, respectively. Although fever was the second highest reported symptom among nursing home residents in our study, only 28.1% reported a fever during their COVID-19 episode. In contrast to these earlier findings [24], our nursing home residents reported less coughing (18.1% in PICOV compared to 62.7% by Neumann-Podczaska). The reasons for these differences are not clear, but could firstly be attributed to memory recall errors as this data is collected retrospectively [25]. Secondly, the study population is different, as our data includes only current residents at nursing homes that survived their SARS-CoV-2 infection. This is in contrast with the systematic review [24], where half of the participants were hospitalised and a fifth of the total population died. Finally, our findings are more in line with findings from care homes in the UK [26], as the authors report a more comparable proportion of fever and coughing.
More than half (56.6%) of the nursing home staff members with a past SARS-CoV-2 infection reported to be fatigued during their COVID-19 episode, which makes this the most prevalent symptom. This incidence is in line with studies in Italian (47.6%) and Swedish (65.0%) healthcare workers (HCW) in hospitals [27,28]. The prevalence of most of our symptoms are consistent with the observations made by Rudberg et al. [28]. Moreover, while a quarter of the HCW in Sweden [28] and nearly a third of the Italian HCW [27] documented dyspnoea as a symptom, 27.8% of our staff members mentioned dyspnoea during their COVID-19 illness. Although the prevalence of symptoms in the present cohort is comparable to those observed in HCW, we should note some key differences between these populations. First and foremost, both the Swedish and Italian study investigated HCW in hospitals, which is fundamentally different from a nursing home setting. Moreover, we enrolled fewer nurses and physicians and more nonmedical support staff such as administrative, support, and kitchen staff.
In the PICOV cohort, asymptomatic SARS-CoV-2 infections (i.e. received no or negative RT-qPCR nasal swabs and a positive Ab test) occur more frequent in residents than in staff members. This is in line with recent observations by Mori et al. [3], who noted three times as many asymptomatic cases in older people (60 years and over) than in young people (18 -60 years old) with a confirmed SARS-CoV-2 infection. This is corroborated by the symptom data, as residents systematically reported fewer symptoms than staff members in PICOV. Another symptom that was reported less frequently among the geriatric population as compared to the staff was ageusia and/or anosmia. This is in agreement with previous findings [29,30] and could be attributed to normal ageing, as taste and smell tend to subside with age [31].
A third of the staff members reported persistent symptoms at baseline, with fatigue, ageusia and/or anosmia, and dyspnoea being the most prevalent, which corroborates previous findings [32,33]. In contrast, only a tenth of the residents indicated any persistent symptom at baseline. Most of them indicated to have a cough, fatigue, or dyspnoea. In both residents and staff members, fatigue was still present a few months after their SARS-CoV-2 infection. In 2009, a follow-up study of 233 SARS survivors in Hong Kong observed that 40% of the responders had a chronic fatigue problem at their four-year follow-up [34]. Interestingly, the geriatric population reported fewer symptoms during their SARS-CoV-2 infection and fewer persisting symptoms 1-10 months afterwards. In summary, nearly three quarters of the PICOV participants indicated to have no more nor additional symptoms one to ten months after symptom onset, which is in line with other non-hospitalised COVID-19 individuals [35].
Our study has several strengths. Firstly, we recruited a representative sample of both staff members and residents of nursing homes across Belgium. As such, our research population includes participants 18 to 102 years old, providing a broader range of ages compared to other published studies. Secondly, the participants are followed closely and sampled frequently in the case of ILI or ARI, allowing us to capture the development of SARS-CoV-2 Ab already early on during an infection. Thirdly, additional samples are collected at the various sampling moments, which allows for additional measurements for immune response monitoring.
We also acknowledge some limitations. First of all, the symptom registration of the COVID-19 episode occurs one to ten months after symptom onset and is therefore subjected to recall bias [36]. Nevertheless the authors proposed that patients tend to recall symptoms better than health-related quality of life. Another shortcoming is that we are unable to capture those individuals with asymptomatic SARS-CoV-2 infections for the duration of the PICOV study with our case definition.

Conclusion
Nursing home residents (both with and without a prior SARS-CoV-2 infection) systematically reported fewer symptoms than staff members. Moreover, results from prior nasopharyngeal RT-qPCR and baseline serology show that antibody development after a SARS-CoV-2 infection differs between residents and staff members. As such, we can postulate that disease development and symptoms are different between a geriatric and younger population. Therefore, the influence of a prior SARS-CoV-2 infection on the occurrence and severity of a future ILI and/or ARI might differ between residents and staff.

Declarations
Ethics approval and consent to participate The study was approved by the Ethics Committee of the Hôpital Erasme in Brussels, Belgium (reference: P2020/424/ B4062020000134) and is registered at ClinicalTrials.gov (reference: NCT04527614).

Consent for publication
Not applicable.

Availability of data and material
The data sets analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.
Funding MEG obtained funding from The Belgian Federal Government.
Author's contributions MEG conceptualised, designed, and coordinates the PICOV-study, and wrote the first draft of the manuscript. KYN helped to draft the manuscript and performed the statistical analyses and data visualisation. PP helped to draft the manuscript and aided in the data visualisation and coordination of the study. CB, IT, and ID performed the RT-qPCR measurements and helped design the study. MNS, MV, and NVL helped in the design and conceptualisation of PICOV, and are responsible for the study nurses. KKA, AM, and SG were also involved in the study design, and performed PBMC isolations. All authors critically reviewed and approved the manuscript.