Seroprevalence of Antibodies against SARS-CoV-2 Infection among Healthcare Workers at a Tertiary Hospital in Saudi Arabia

Background The nature of the healthcare workers' jobs standing at the frontline against the coronavirus disease 2019 (COVID-19) puts them at a higher risk of unknowingly contracting the disease and potentially contributing to the spread. This study aims to assess the overall positive seroconversion prevalence of SARS-CoV-2. Methods This is a longitudinal cohort study of healthcare workers at a tertiary hospital serving patients in several districts in the Eastern Province of Saudi Arabia. Participants were recruited between June - December 2020. Each participant had a serology blood test and completed the World Health Organization's risk factors assessment questionnaire. with P=0.03, and alcohol-based or and after and PPE The that those were less likely wear PPE and to perform proper hand The practice of to reduce infection 30 . Multiple interventions were used to promote hand even before the current pandemic 30,31,32 . Staff compliance with the hospital's robust Infection


Introduction
Since the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the entire world had been threatened 1 . As of September 2021, nearly 229 million cases of coronavirus disease 2019  have been reported with more than 4.7 million deaths worldwide 2 . The pandemic has affected the healthcare systems and resources severely in many countries, especially with the healthcare workers (HCWs) are in the highest-risk group due to the nature of their job and increased exposure while closely interacting with infected individuals 3 .
There is a level of uncertainty of epidemiological, clinical, and virological characteristics associated with a novel virus and its severity and ability to spread amongst humans. With SARS-CoV-2 high transmission rate, the disease caused a tremendous burden on the healthcare systems and frontline HCWs 1 . As a result of the disease's nature of transmission and the HCWs high risk of infection, they may have unknowingly contracted the disease and potentially contributed to the spread 4 . Several studies have reported that the evidence of COVID-19 infection among HCWs is growing with a current rate of (2.3%) in Saudi Arabia 5 and up to (17.4%) globally 4 . A number of these studies also reported that (38%-48%) of the HCWs with positive seroconversion never experienced any symptoms 6,7 .
Questions have been risen about using reverse transcription-polymerase chain reaction (rt-PCR) solely for infection screening as there is still little to be known about the SARS-CoV-2 virus and the knowledge of asymptomatic infections among HCWs is also limited 8 . Therefore, serological testing can provide information on the proportion of individuals who have been infected with COVID-19. It is crucial to monitor the prevalence of infection in critical subgroups of the population such as HCWs and nursing homes employees 9 . This is bene cial for many vital reasons, for one, it will help determine risks and levels of exposure among staff and identify high-risk departments/ wards. Moreover, information about current and previous infections can be useful for healthcare resource planning as well as avoiding unnecessary quarantines for the staff 9 .
Healthcare workers are considered our shield against COVID-19, the nature of their job has placed them on the frontline to ght this pandemic globally placing them at the highest risk to contract an infection which might lead to a heavier burden on healthcare organizations 10 . A better understanding of the pattern and prevalence of the SARS-CoV-2 infection can guide HCWs for better protection as well as assistance to policymakers to develop policies and regulations for infection prevention and control in the clinical setting 10 . Therefore, it is crucial to understand and determine the correlation between the positive seroconversion and duration of immunity as well as the possibilities of reinfection amongst HCWs 9 .
This study aims to assess the immune status of HCWs at a tertiary hospital. We aim to assess the overall positive seroconversion prevalence to SARS-CoV-2 as well as the prevalence of asymptomatic infections. Moreover, we aim to assess the risk factors of seroconversion amongst HCWs using the World Health Organization's (WHO) protocol for assessment of potential risk factors for 2019-novel coronavirus (2019-nCoV) infection among healthcare workers in a healthcare setting as one of their Early Investigations protocols 11 .

Study design
The study received the hospital's Institutional Review Board's approval (IRB # 20-09) on the 13th of May 2020. This is a longitudinal cohort study of healthcare workers at a tertiary hospital located in Dhahran serving patients in several districts in the Eastern Province of Saudi Arabia. The study took place from June 2020 until the end of April 2021.
Each participant was assigned a study ID and written informed consent was obtained as well as a hard copy of the WHO's risk factors assessment questionnaire.

Study Population and Sample
Initially, the participants were randomly selected from the hospital's employees' database and invited to participate in the study via emails. However, due to the slow recruitment, an open invitation was sent to all staff members through staff announcement to their emails. Recruitment was completed by December 2020 and follow-up appointments were carried on until April 2021. Participants were asked to visit the research team in 2 months period for a follow-up test. Participants with positive SARS-CoV-2 IgG assay were asked to return to the research team for a third follow-up visit. As of December 2020, our hospital had 3939 staff members and 383 housekeepers working in all departments. This study included 682 participants working in different departments at the hospital from front-liners dealing with COVID-19 patients on daily basis to administrative whom had minimal contact with any patient.

Data Collection
Blood samples were collected from participants by a phlebotomist at the local hospital laboratory. The SARS-CoV-2 IgG assay results were then sent to the research team as positive or negative and their index which then entered into the study database.
The WHO Questionnaires extracted from their protocol for assessment of potential risk factors for 2019novel coronavirus (2019-nCoV) were administered to all participants upon consenting to the study and before their blood collection appointments. All completed questionnaires were entered into the study database by two nurses and validated by the study's principal investigator.
ARCHITECT SARS-CoV-2 IgG Assay ARCHITECT SARS-CoV-2 IgG Assay was used throughout the whole study. The assay is an automated, two-step immunoassay for the qualitative detection of IgG antibodies to SARS-CoV-2 in human serum using chemiluminescent microparticle immunoassay (CMIA) technology.
The index reference range interpretation for this qualitative IgG test is positive if the index was 1.4 or above and negative if the index was below 1.4.

Validation of SARS-CoV-2 IgG Testing
The validation study of the SARS-CoV-2 IgG test was carried on Architect i2000SR System. Precision study with in-run and total is not applicable as this is a qualitative method. Positive and negative controls for SARS-CoV-2 IgG Assay were run daily for fteen days and it has been indicated acceptable result. A total of 23 patient serum specimens was tested on the Architect i2000SR System, and at the same time on the Architect i2000SR in King Fahad Specialist Hospital for SARS-CoV-2 IgG test, results compared for parallel testing. The comparison study showed that the results of the SARS-CoV-2 IgG on the DH-Architect i2000SR System and KFSH-Architect i2000SR System were compared and correlated. A comparison study indicated 100% speci city and sensitivity. The validation study concluded that the SARS-CoV-2 IgG test performed on Architect i2000SR System is acceptable for patient testing.

Summary and explanation of the test
The assay is designed to detect IgG antibodies to the nucleocapsid protein of SARS-CoV-2 in serum from patients with signs and symptoms of infection who are suspected of coronavirus disease (COVID- 19) or in serum of subjects that may have been infected by SARS-CoV-2. The SARS-CoV-2 IgG assay can be used as an indication of a possible recent infection. However, negative results do not rule out previous infections, especially for those who have been in contact with positive cases.
The incubation period of COVID-19 ranges between 1 and 14 days. The host immune system reacts to the infection by SARS-CoV-2 by producing speci c antibodies. These antibodies have been reported to appear in serum or plasma of infected individuals after the detection of viral ribonucleic acid (RNA) in swabs and a few days to 2 weeks after the onset of symptoms. Speci c IgG antibodies to SARS-CoV-2 are detectable during the symptomatic phase of the disease after RNA is no longer detectable.
The sensitivity of combining RNA with antibody results has been reported as above 99%. The persistence of IgG antibodies allows identi cation of people who have been infected in the past, recovered from the illness, and possibly become immune.

Statistical Analysis
The distribution of quantitative values of variables for the subjects has been examined with descriptive statistics (such as Mean, and Standard deviation). The distribution of all-qualitative (i.e. close-ended) values of variables for both demographic and research variables of the study samples have been examined with frequency tables. The association tables have been calculated by using the chi-square test with cross-tabulation. A P-value < 0.05 will be considered for statistical signi cance. Binary logistic regression has been used to predict the odds of the cases based on the independent variables which were the reported symptoms with respect to the dependent variables such as previous infection of COVID-19 and positive seroconversion. The statistical analysis has been done using SPSS (Statistical Package for Social Sciences) Package with version 25.

Results
A total of 682 HCWs consented to participate in the study. The overall participation rate was (15.7%) from all the hospital staff. Out of the 682 participants, (52.8%) were females, (39%) were Saudi nationals, (88.2%) were non-smoker, and (56.8%) of the participants were clinicians (physicians and nurses), following that 26.3% are from supporting services departments (such as patient safety and quality improvement, maintenance, population health, and housekeeping), and nally 16.9% were from the applied medicine departments (such as rehabilitation, laboratory, and pharmacy). The mean age of the participants was 44.5 (±10) and the mean years of professional experience 16 (±10) years (Table 1).
One hundred and twelve (15.2%) participants had a positive SARS-CoV-2 rt-PCR before taking part in the study. However, only 87 tested positive for SARS-CoV-2 antibodies, a prevalence of (12.7%) of all participants. Out of the 87 positives for SARS-CoV-2 antibodies, 17 participants never tested positive for COVID-19 rt-PCR, a prevalence of (2.9%) of all participants who never tested positive for COVID-19 rt-PCR (Table 1).
Two factors were found statistically signi cant in the infection prevention and control measures to having positive SARS-CoV-2 IgG assay. The rst factor was using alcohol-based hand rub or soap and water after the risk of body uid exposure with (P=0.02, CI: 95%), and the second factor was wearing personal protective equipment (PPE) when indicated with (P=0.03, CI: 95%) ( Table 2). In addition, exposure to the community such as delivery personnel, and/or family visits had no statistical signi cance difference between positive and negative SARS-CoV-2 IgG assay (P> 0.05, CI: 95%), however, exposure to co-workers had a statistical signi cance to testing positive for COVID-19 rt-PCR with (P= 0.01, CI: 95%).
In the regression analysis, fever was the only common symptom with statistical signi cance to be reported by both participants who had tested positive for COVID-19 rt-PCR and SARS-CoV-2 IgG assay. Moreover, it was also found that it is (33%) likely that participants with positive COVID-19 rt-PCR would suffer from cough, (24%) are likely to report headaches, (22%) are likely to feel fatigued, and it is also most likely that 5 out of 10 would suffer from conjunctivitis (Table 5). However, no signi cance other than fever was found in the regression between positive SARS-CoV-2 IgG assay and symptoms presentation.     25 and similar symptomatology in other studies 23,26,27,28 .
In 2020, a hospital in China conducted a study of obesity and its association with COVID-19 severity 29 .
The study concluded that patients who suffer from obesity have increased odds of developing and progressing to severe COVID-19 symptoms 29 . In this study, our ndings showed that obesity is signi cantly associated with many COVID-19 symptoms as well more than any other comorbidity such as diabetes.
Elucidating factors associated with positive SARS-CoV-2 serology revealed two associated factors with positivity in bivariate analysis. These are always using alcohol-based hand rub or soap and water after (risk of) body uid exposure and always wearing PPE when indicated. The data showed that those staff who were positive were less likely to wear PPE and to perform proper hand hygiene. The practice of hand hygiene is of paramount activity to reduce infection 30 . Multiple interventions were used to promote hand hygiene even before the current pandemic 30,31,32 . Staff compliance with the hospital's robust Infection Prevention and Control program and guidance helped in maintaining the risk of infection and prevalence of SARS-CoV-2 amongst HCWs considerably low compared to other national and international healthcare organizations.
During the pandemic, isolation of suspected patients, the use of facemasks, and intensi ed hand hygiene were important for the prevention of nosocomial transmission of COVID-19 33 . One study showed that multiple services were contaminated and had positive SARS-CoV-2 RNA 34 . Another study from KSA showed high knowledge and practice scores in relation to hand hygiene and the use of masks 35 . In a case-control study, frequent handwashing, social distancing, and avoidance of close contact were independently associated with a lower risk for SARS-CoV-2 infection 36 .
One of the main limitations we exhibited in this study was the inability to categorize our participants into groups according to their level of exposure whether it was high or low to the virus / COVID-19 patients as this information was not comprehensively provided by the participants. In addition, due to the slow accrual, the study protocol was amended from randomized selection to open invitation which eventually resulted in achieving the targeted sample size (more than 10% of the hospital staff) at the expense of risk of selection bias. Finally, this study was conducted prospectively, participants depended fully on their memory and personal interpretation of their symptoms and association to the infection.
This study was carried out during the rst wave of COVID-19 and before the availability of vaccination. Our ndings showed that the positive seroconversion rate was considerably low amongst our healthcare workers and similar to other national and international healthcare organizations. Although the results of the study can be interpreted as a success in following the recommendations of the Intervention Prevention and Control Division, seropositivity correlated signi cantly with two factors of infection prevention which were appropriately alcohol-based hand rub or soap and water after the risk of body uid exposure and wearing personal protective equipment when indicated Declarations