COVID-19 Seroprevalence Test for IgG Antibody Levels Among Healthy Donors Across Different Pandemic Phases in Jeddah

Summary

This study assessed COVID-19 seroprevalence in Jeddah, Saudi Arabia, among healthy blood donors across pre-lockdown, during-lockdown, and post-lockdown periods. Of 3,825 samples, 4.5% tested positive for IgG antibodies. Seroprevalence increased significantly post-lockdown, highlighting the role of asymptomatic transmission and the effectiveness of initial restrictions.

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has significantly impacted global public health and the economy. This study aimed to investigate the seroprevalence of IgG antibodies against SARS-CoV-2 among healthy blood donors in Jeddah, Saudi Arabia, to better understand the dynamics of COVID-19 transmission during three distinct periods: pre-lockdown, during-lockdown, and post-lockdown. The seroprevalence was assessed concerning demographic factors such as gender, age, blood group, and nationality. A cross-sectional study was conducted from December 2019 to December 2020. A total of 3,825 blood samples from healthy donors were screened using an in-house enzyme-linked immunosorbent assay (ELISA) for anti-SARS-CoV-2 IgG antibodies detection. Positive samples were further confirmed using a micro-neutralization (MN) assay. Of 3,825 serum samples, 173 (4.5%) were tested positive by ELISA, and 147 samples were confirmed positive by MN. All samples collected before the lockdown (December 2019 to February 2020) were negative for IgG antibodies. During the lockdown period (March 2020 to June 2020), 44 out of 1,482 samples (2.97%) were tested positive. After the lockdown was eased (July 2020 to December 2020), the positivity rate increased to 12.15% (103 out of 848 samples). The overall seroprevalence rate among healthy blood donors was 3.84%, with significantly higher rates among non-Saudi and male donors (p = 0.00). The findings suggest that COVID-19 restrictions implemented by the Saudi authorities were effective in reducing the initial spread of the virus. However, the marked increase in seroprevalence over time suggests that asymptomatic or mildly symptomatic individuals may pose a significant risk for continued transmission. Continuous monitoring of seroprevalence is essential to control the spread of COVID-19.

Introduction

Coronavirus disease 2019 (COVID-19), caused by the novel beta-coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a major global health concern due to its devastating outcomes¹. The virus is thought to have originated from genetic recombination between bats and pangolins². The virus was subsequently transmitted to humans, demonstrating efficient human-to-human transmission through aerosols. The first case of COVID-19 was reported on 31 December 2019 in Wuhan, China³. The virus quickly spread across China and subsequently to the rest of the world⁴.

The World Health Organization declared SARS-CoV-2 a pandemic on March 11, 2020, with more than 400 million cases reported worldwide in 2022, with 6 million deaths⁵. The cumulative number of COVID-19 cases reported to WHO has reached more than 776 million worldwide and more than 841,000 in Saudi Arabia⁶. The number may be much higher because people infected with the disease are asymptomatic or have mild to moderate symptoms, although COVID-19 can cause severe complications such as pneumonia, acute respiratory distress syndrome, and organ failure⁷.

This pandemic has affected the entire world; governments and healthcare organizations have been quick to take strict measures since day 1 to limit the spread of the novel coronavirus. These measures include identifying and quarantining suspected and confirmed cases, establishing isolation units, social distancing, widespread use of face masks, and travel restrictions⁸. Saudi Arabia was also among the first countries to impose strict measures after reporting the first case of novel coronavirus on March 2, 2020. The Ministry of Health and policymakers in Saudi Arabia imposed strict control measures in February 2020, such as closing mosques, closing schools and universities, providing free healthcare to all COVID-19 patients⁹, evacuating Saudis from affected countries, suspending domestic and international flights, and suspending tourism and Umrah¹⁰. These restrictions were gradually eased after May 28, 2020, through a three-phase plan that maintained key measures such as social distancing, face masks, and virtual learning. On December 17, 2020, the Pfizer-BioNTech COVID-19 vaccine became available in Saudi Arabia¹¹.

Multiple studies investigated COVID-19 seroprevalence to identify the true number of asymptomatic infections, which may contribute significantly to disease transmission^12,13. Such studies are essential for evaluating herd immunity, an important indicator of virus transmission within populations. Identifying asymptomatic COVID-19 cases is also crucial for preventing and controlling the spread of the virus. Effective control of COVID-19 transmission requires efforts to detect asymptomatic and mildly symptomatic cases who do not seek healthcare and remain undetected by surveillance systems¹⁴. Understanding the actual number of infected individuals in a community is essential for estimating the need for measures such as hospitalization rates¹⁵. Furthermore, seroprevalence data can help expand knowledge on transmission dynamics and inform suitable control measures, such as social distancing, educational institution closures, and vaccination.

Several studies have explored the antibody response to SARS-CoV-2 infection, considering various factors such as demographics (age, sex, and ethnicity). However, many of these studies have yielded inconsistent or inconclusive results, partly due to differences in study design, strategies, and outcome measures. In particular, while most studies found no correlation between antibody response and age or gender¹⁶, some reported conflicting findings that lacked robust statistical analysis. Also, there is also a lack of published data explaining immunological responses across different ethnic groups.

Previous research aimed at identifying the true number of asymptomatic individuals has been conducted on approximately one million blood donors in 20 countries from January 2020 to January 2021. By reviewing thirty-three of these studies, it observed the seroprevalence was less than 10% in 79% of the studies as of December 2020, indicating low herd immunity¹⁷. One study conducted in Saudi Arabia, in Al-Madinah, had reported a high prevalence of SARS-CoV-2 antibodies among donors¹⁸. Although the study had several limitations, including the absence of confirmatory testing with a standard assay¹⁹, it provides an important basis for understanding antibody prevalence and underscores the need for further confirmatory research in this area.

Further investigation was needed to assess the seroprevalence of SARS-CoV-2 across various demographics in multiple cities over extended periods. Given its diverse population, Jeddah-Saudi Arabia's largest city after Riyadh-was of particular interest as a study area. Therefore, our study was expanded beyond its initial scope to include a larger sample size and a more comprehensive analysis of seroprevalence trends across three distinct phases: before, during, and after the lockdown. These expanded efforts aimed to provide a clearer understanding of COVID-19 transmission patterns and the role of blood donors in reflecting community-level antibody prevalence, ultimately guiding future public health strategies.

The significance of this study lies in its contribution to understanding seroprevalence trends among blood donors during three distinct pandemic phases: pre-lockdown, during lockdown, and post-lockdown. Blood donors represent a unique population for such studies due to their generally healthy status and frequent contributions, which can provide valuable insights into asymptomatic or mildly symptomatic infections²⁰. Understanding seroprevalence in this group is crucial for assessing community-level exposure to the virus and its implications for public health planning, including vaccination strategies.

This study hypothesizes that seroprevalence among blood donors will show a marked increase following the easing of lockdown measures, reflecting the delayed spread of SARS-CoV-2 after restrictions were lifted. By comparing pre- and post-lockdown seroprevalence, the research aims to provide insights into the dynamics of COVID-19 transmission and the effectiveness of initial containment measures. Furthermore, this study seeks to explore demographic factors such as gender, age, and nationality to understand their influence on seroprevalence. This research aimed to investigate the seroprevalence of IgG anti-SARS-CoV-2 among BDs from December 2019 to December 2020 at KAUH, Jeddah. The study had three specific objectives: Firstly, to measure the prevalence of IgG antibodies to SARS-CoV-2 in the general population and correlate it with demographic data such as gender, age, blood group, and nationality. Secondly, to determine whether case zero or any early cases were present before the first officially reported case in the country on 2nd March 2020. Thirdly, investigating the dynamics of COVID-19 infection at different time points (before, during, and after the lockdown period).

Protocol

This research was approved by the Research Ethics Committee (REC) of the Unit of Biomedical Ethics at the Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia (Ref. No. 219-24). Informed consent was obtained from all participants for this study.

1. Study design

NOTE: A cross-sectional study at the Special Infectious Agents Unit (SIAU), King Fahd Medical Research Center (KFMRC), Jeddah, Saudi Arabia, to analyze COVID-19 prevalence in voluntary blood donors (BDs) from December 2019 to December 2020 was conducted.

1. For an overview of the study design, highlighting the pandemic phases, participant recruitment, and the data collection and analysis methodology, see Figure 1.
2. Use a serological immunoassay to detect anti-SARS-CoV-2 IgG antibodies, adhering to institutional ethical guidelines.

2. Sample collection

1. Use the following inclusion criteria: Healthy adults aged 18-60 years; Willingness to provide informed consent for participation in the study; No history of SARS-CoV-2 infection before participation.
2. Use the following exclusion criteria: Individuals with a known history of COVID-19 infection; Recent illnesses or flu-like symptoms; Individuals who have had contact with a confirmed case of COVID-19 within the last 14 days; Pregnant or breastfeeding women; Individuals with insufficient sample volume.
3. Collect whole blood samples (3-5 mL) from healthy, eligible blood donors. In this study, the blood samples were collected in yellow and red top plain collection tubes at King Abdulaziz University Hospital. We coordinated with the blood donation department to ensure samples were obtained from all eligible donors. These samples were then sent to the laboratory for further analysis.
4. Record demographic data, including age, sex, blood group, and nationality, from the KAUH Transfusion Services donation area for all participating donors.
   NOTE: In this study, 3,825 blood samples were collected from donors at King Abdulaziz University Hospital (KAUH), Jeddah, between December 2019 and December 2020.
5. Conduct PCR medical examinations to confirm that participants are free from recent illnesses, particularly flu-like symptoms or recent contact with individuals suspected or confirmed to have COVID-19. Exclude any donors with a positive medical history of SARS-CoV-2 infection, duplicated samples, those with insufficient volume, and unlabeled samples.
   NOTE: Data can be paused after collection and stored at -20 °C until ready for processing.

3. Serum processing

1. Centrifuge blood samples at 200 x g for 10 min to obtain serum (supernatant). Store the serum at -20 °C in a Biosafety Level 3 facility at SIAU until testing.
   CAUTION: Ensure proper handling of samples in a Biosafety Level 3 facility.

4. In-house enzyme-linked immunosorbent assay (ELISA)

1. Screen sera samples obtained from healthy blood donors (BDs) for anti-SARS-CoV-2 IgG antibodies using a validated and optimized in-house ELISA¹⁹.
2. Coat the microtiter plates with 100 ng of SARS-CoV-2 spike recombinant protein diluted in PBS. Incubate the plates overnight at 4 °C.
3. Wash the plates 3x with phosphate-buffered saline with Tween 20 (PBST). Block the plates by adding 5% skim milk for 1 h at room temperature. Wash the plates 3x with PBST after blocking.
4. Dilute sera samples at 1:100 in 5% skimmed milk. Add 100 µL of diluted sera samples to each well and incubate for 1 h at 37 °C.
5. Wash the plates 3x with PBST. Add 100 µL of goat KPL peroxidase-labeled antibodies to human IgG (diluted 1:64,000 in PBST) to each well and incubate for 1 h at 37 °C. Wash the plates 3x with PBST after incubation.
6. Add 100 µL of 3,3',5,5'-Tetramethylbenzidine (TMB) to each well and incubate for 5 min for color development. Add 100 µL of hydrochloric acid to stop the reaction.
7. Measure the absorbance at 450 nm using a microplate spectrophotometer. Run all samples in duplicate and use samples from COVID-19-recovered patients as positive controls.
8. Consider a specimen reactive (positive) for anti-SARS-CoV-2 IgG antibodies if the optical density (OD 450) value is equal to or greater than 0.27. Consider a specimen non-reactive (negative) if the OD450 value is less than 0.27.
9. Subject positive samples to the micro-neutralization assay (MN) as the gold standard for confirmation²¹.

5. Micro-neutralization (MN) assay

NOTE: Conduct all procedures in a Biosafety Level 3 (BSL-3) facility to ensure appropriate handling of infectious agents.

1. Assess the presence of virus-specific antibodies and measure their neutralization using the MN assay, considered the gold standard method²¹.
2. Heat-inactivate serum samples by placing them in a water bath at 56 °C for 30 min. Prepare serial dilutions of heat-inactivated serum samples at 1:10, 1:20, and 1:40 using Dulbecco's Modified Eagle Medium (DMEM) containing 100 mL from 50% tissue culture infection dose (TCID 50) of SARS-CoV-2.
3. Culture Vero E6 cells in DMEM a starting cell number of 2-3 x 10⁵ cells/well to reach 70%-80% confluency in 24 h. Once confluent, add 100 µL of the diluted serum samples to the wells containing the confluent Vero E6 cells.
4. Add 100 µL of DMEM containing 50% tissue culture infection dose (TCID50) of SARS-CoV-2 to each well. Incubate the plates at 37 °C in a 5% CO₂ humidified incubator for 3-4 days.
5. Assess the cytopathic effect (CPE) 3 days after infection using an inverted microscope to read the plate. Determine MN titers as the highest dilution of the serum sample that completely prevents the viral-induced cytopathic effect. MN titer ≥ 1:20 is considered positive for the presence of virus-specific antibodies.
6. Include wells with SARS-CoV-2-infected cells without human serum as positive controls. Include wells without virus or serum as negative controls.

6. Statistical analysis

1. Use a spreadsheet to organize clinical and diagnostic demographic data for all donor participants and generate charts and graphs for test results.
2. Perform statistical analysis using SPSS software to calculate the percentage of variables.
   1. Present categorical variables such as age group, gender, blood group, and nationality as numbers (n) and percentages (%).
   2. Present continuous variables, such as donor age, as mean ± standard deviation (SD).
3. Conduct a Chi-square test to examine the association between demographic factors and seroprevalence results.
   1. If an association is detected, apply a post-hoc Bonferroni test to determine the significant factor.
4. Define statistical significance as a p-value of less than 0.05.

Results

The socio-demographic characteristics of blood donor participants were analyzed from 3,825 blood samples collected at King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia, between December 2019 and December 2020, to evaluate the seroprevalence of COVID-19 antibodies. This period was divided into three distinct phases: pre-lockdown (December 2019 to February 2020), during lockdown (March 2020 to June 2020), and post-lockdown (July 2020 to December 2020). The number of blood donors varied across these phases: 1,...

Discussion

In response to the COVID-19 pandemic, many countries, including Saudi Arabia, have imposed a variety of control and restriction measures^9,23. The impact of these test measures on reducing virus transmission is unknown, and further investigation is needed²⁴. In this study, the prevalence of SARS-CoV-2 in Jeddah was assessed during three consecutive periods of the COVID-19 pandemic (before, during, and after the lockdown period). The results...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
African green monkey kidney cells Vero E6	ATCC	CRL1586	Cells used for MN assay
Difco Skim Milk BD Biosciences (5% skim milk with PBST)	Becton-Dickinson & Company	232100	Blocks non-specific binding
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco by Life Technologies	2241121	Growth medium for Vero E6 cells
Elx 800 Bioelisa Reader	Biokit	185681	Measures absorbance at 450 nm in ELISA assay
Hydrochloric acid standard solution (HCL)	Sigma-Aldrich	7647-01-0	Stops the color development reaction in ELISA
Immuno-plate (96-well flat plate)	SPL Life Sciences	32296	Used as the surface for ELISA assay reactions
Incubator 37 °C	LabTech	LIB–080M	Incubator for ELISA plate
Phosphate buffer saline (PBS)	Sigma-Aldrich	79382	Used for sample dilution and washing
SARS-CoV-2 B.1.617.2 Spike S1+S2 trimer Protein (ECD, His tag) (SARS-CoV-2 full-length spike protein)	Sino Biological	40589-V08B1	Used for coating microtiter plates in ELISA assay to detect SARS-CoV-2 antibodies
SARS-CoV-2 clinical isolate	SIAU	NA	Virus used for micro-neutralization assay
Secondary Ab (goat KPL peroxidase-labelled antibodies to human IgG)	Sera care	5220-0277	Secondary antibody to bind and detect human IgG in ELISA
SureBlue TMB 1-Component Microwell Peroxidase Substrate	Sera care	5120-0075	It is a substrate solution used to bind with secondary antibody and then change the color of the reaction in the ELISA plate
Tween 20 Surfact -Amps Detergent Solution	Thermo-Fisher Scientific	85113	Adding tween to PBS buffer to make (PBST) as detergent to prevent non-specific binding by washing