323J Contemp Med Sci | Vol. 8, No. 5, September-October 2022: 323–326 Original A Study of Factors that Impact the Production of Anti-SARS-CoV-2 Antibodies in Patients with Covid-19 Nawfal R Hussein1, Amer Balatay2, Ameen M Mohammad3, Kuldeep Dhama4, Narin A Rasheed5* 1Department of Biomedical Sciences, College of Medicine, University of Zakho, Zahko, Kurdistan Region, Iraq. 2Department of Clinical Pharmacy, College of Pharmacy, University of Duhok, Duhok, Kurdistan Region, Iraq. 3Department of Internal Medicine, College of Medicine, University of Duhok, Duhok, Kurdistan Region, Iraq. 4Division of Pathology, ICAR-Indian Veterinary Research Institute (IVRI), Izatnagar- 243122, Bareilly, Uttar Pradesh, India. 5Department of Medical Laboratory Technology, College of Health and Medical Technology-Shekhan, Duhok Polytechnic University, Duhok, Kurdistan Region, Iraq. *Correspondence to: Narin A Rasheed (E-mail: narin.rasheed@gmail.com) (Submitted: 29 April 2022 – Revised version received: 12 May 2022 – Accepted: 17 May 2022 – Published Online: 26 October 2022) Abstract Objectives: The aim of this paper was to investigate the impact of different variables on the production of antibodies in patients who were infected with Covid-19. Methods: This cross-sectional study was conducted in Duhok City, Kurdistan Region of Iraq. The study was conducted between January 2021 and March 2022. Demographic data were collected via face-to-face interview. Antibody levels were determined using Elecsys Anti-SARS-CoV-2. Results: The levels of antibody were studied in 138 patients. A significant association was found between antibody levels and the age of the participants (r = 0.175; P = 0.04). Besides, a significant correlation was found between antibody levels and the duration of symptoms (r = 0.206; P = 0.015). The antibody levels were not associated with gender; history of chronic diseases; marital status or time interval before testing. Conclusion: Different variables that may impact the levels of antibody were studied. Significant associations were found between antibody levels and both age and duration of symptoms. Our results can be used by healthcare providers to focus on patients who are at risk of low antibody production. Keywords: SARS-CoV-2, antibodies, Covid-19, Duhok, Iraq ISSN 2413-0516 Introduction COVID-19 caused by SARS-CoV-2 that was discovered in Wuhan, China, in December 2019 and rapidly spread world- wide.1 Since the discovery of first cases of Covid-19 in Kurd- istan Region of Iraq in March, 2020, the Region passed through three devastating waves with a case fatality rate of 2%. During the first wave, strict measures were taken to con- trol the infection2 and the case fatality rate was low. Then, due to public fatigability and unwillingness,3,4 the measures were relaxed leading a sharp increase in morbidity and mortality of Covid-19 cases. Such an increase in the cases had a negative impact on the already weak health system.5–8 The number of COVID-19 patients exceeded the capacities of acute care beds and home management scheme was launched to manage cases of severe covid. Covid-19 has a wide array of symptoms particularly fever and respiratory symptoms such as cough and shortness of breath.9,10 Non-respiratory symptoms are not uncommon such as gastrointestinal symptoms including diarrhea and vomiting. Herd immunity can be the most important factor to control the pandemic. Herd immunity is defined as the reduction in the number of cases due to the development of immunity by natural infection of vaccina- tion.11,12 The spike (S) protein is the antigenic protein of SARS-CoV-2 that mediate the fusion with human angioten- sin-converting enzyme 2 (ACE2). The S antigen is consisted of two subunits: 1 (S1) and 2 (S2). The latter is involved in the fusion process between the cell membranes and viruses. Anti- bodies that inhibit this specific binding are known as neutral- izing antibody. Such antibodies play a pivotal rule in the prevention of reinfection.13–15 It is worth mentioning that studies have shown that antibody response induced by natural infection wane over time and different factors may impact the production and the intensity of antibody production and reinfection is possible.13–15 The aim of this paper was to inves- tigate the impact of different variables on the production of antibodies in patients who were infected with Covid-19. Materials and Methods Study Design This cross-sectional study was conducted in Duhok City, Kurdistan Region of Iraq. The study was conducted between January 2021 to March 2022. Blood samples were collected form participants who were at least 18 years old, had a history of confirmed covid-19, had not received vaccination and agreed to participate in the study. Then, 5–10 cm3 of venous blood samples were collected using 10-cm3 syringes. The sam- ples were immediately transported to the research center, and sera were separated from the blood and kept frozen at –20°C. Demographic data were collected via face-to-face interview. Anti-SARS-CoV-2 Antibody Antibody levels were determined using Elecsys Anti- SARS-CoV-2 (Roche Diagnostics International Ltd, Rotkreuz, Switzerland), which is an in-vitro immunoassay to determine antibodies (including IgG) to the SARS-CoV-2 spike (S) pro- tein receptor binding domain (RBD) in human serum and plasma. The assay was performed according to the manufac- turer’s instructions. According to the manufacturer, a cutoff index ≥0.8 indicates a positive result. mailto:narin.rasheed@gmail.com 324 J Contemp Med Sci | Vol. 8, No. 5, September-October 2022: 323–326 Factors Impacting Anti-SARS-CoV-2 Antibody Production Original N.R. Hussein et al. Statistics Binary logistic regression was utilized to analyze the associa- tion between antibody levels and dichotomous data. Pairwise Pearson’s correlation was utilized to investigate the relation- ship between antibody levels and continuous variables. All cal- culations were performed using Minitab 20 software. P value of ≤0.05 was considered significant. Ethics The study and all procedures were approved by Ethics and Scientific Committee of the College of Medicine, University of Zakho. The work was carried out in accordance with The Code of Ethics of the World Medical Association (Declara- tion of Helsinki) for experiments involving humans. Written Informed consent was obtained from all participants. Results Blood samples were collected from participants with a pre- vious history of confirmed Covid-19. Demographic data were collected via face-to-face interview. In this study, the mean age of the participants was 38.44 ± 1.17, and (68/138) 49.28% of the participants were female. Among the participants, (111/138) 80.43% were married and (64/138) 46.38% had a history of chronic diseases (Table 1). Binary logistic regression was utilized to analyze the asso- ciation between antibody levels and dichotomous data. Although antibody levels were higher in females than that found in males, no statistically significant association was found between sex and antibody levels (OR = 0.99; CI = 0.99–1.003; P = 0.7) (Table 2). Besides, no significant association was found between the history of chronic diseases and antibody levels (OR = 1.003; CI = 0.998–1.01; P = 0.1) (Table 2). Antibody levels were higher in married than unmar- ried participants, although the association was not statistically significant (OR = 1.007; CI = 0.999–1.013; P = 0.058) (Table 2). Pairwise Pearson’s correlation was utilized to investigate the relationship between antibody levels and age. A significant association was found between antibody levels and the age of the participants (r = 0.175; P = 0.04) (Figure 1). Additionally, Pairwise Pearson’s correlation was used to study the correla- tion between antibody levels and duration of the symptoms during the infection. A significant correlation was found between antibody levels and the duration of symptoms (r = 0.206; P = 0.015) (Figure 2). The correlation between time interval before testing and antibody levels was investigated. No association was found between antibody levels and time interval before testing (r = 0.038; P = 0.66) (Figure 3). Discussion Kurdistan Region of Iraq went through three devastating waves that impacted the already weak health system. Since the appearance of SARS-CoV-2 pandemic, healthcare providers are aiming to controlling the spread of the infection.16,17 The development of herd immunity after natural infection or vac- cination was the aim. However, reinfection and breakthrough infection appeared to be obstacles for controlling the pan- demic by herd immunity.18 Understanding factors associated with waning of the immunity is important for healthcare pro- viders to focus on patients who are at risk of low antibody pro- duction.19 Therefore; we aimed at studying different variables associated with antibody levels in patients who were infected with SARS-CoV-2. In our study, we found that older age was associated with higher levels of anti-SARS-CoV-2 antibodies. Our results are in agreement with previous studies that found higher level of antibodies in older patients.20–22 This might be explained by that older patients are susceptible to severe infec- tion which might induce more rigorous immune reaction. More studies are needed to investigate the dynamic of anti- body response over the time in older patients. Additionally, it was previously proposed that biological sex impacts immune responses and COVID-19 outcomes.23 In support of this in a Table 1. Characteristics of participants Dichotomous variables No. % Gender Female 68 49.28 Male 70 50.72 Chronic Disease No 74 53.62 Yes 64 46.38 Marital status No 27 19.57 Yes 111 80.43 Continuous variables Mean SE Mean Minimum Maximum Age (year) 38.44 1.17 18 76 Time interval before testing (day) 148.82 4.39 38 313 Duration of symptoms (day) 12.568 0.743 2 45 SE, Standard error. Table 2. Associations between antibody levels and different factors Variables No. Mean SE Mean Minimum Q1 Median Q3 Maximum OR CI P value Sex Female 68 71.95 8.19 0.1 10.22 42.45 131.42 203.6 0.999 0.99-1.003 0.7 Male 70 67.58 8.5 0.1 5.22 40.25 121.9 221.9 Chronic disease No 74 62.04 7.89 0.1 2.82 22.65 114.08 200.3 1.003 0.998-1.01 0.1 Yes 64 78.62 8.76 0.1 10.2 65.55 135.08 221.9 Marital status No 27 46.7 12.3 0.1 1.2 10.9 92 221.9 1.007 0.999-1.013 0.058 Yes 111 75.32 6.59 0.1 7.4 56.7 134.4 203.6 SE, Standard error; Q, Quartile; OR, Odd ratio; CI, Confidence interval. 325J Contemp Med Sci | Vol. 8, No. 5, September-October 2022: 323–326 N.R. Hussein et al. Original Factors impacting anti-SARS-CoV-2 antibody production Fig. 3 Scatterplot showing the association between antibody levels and time interval before testing. No significant associa- tion was found between antibody levels and the interval before testing (r = 0.038; P = 0.66). Fig. 1 Scatterplot showing the association between antibody levels and age of the participants. A significant association was found between antibody levels and age of participants (r = 0.175; P = 0.04) Fig. 2 Scatterplot showing the association between antibody levels and the duration of symptoms. A significant association was found between antibody levels and the duration of symptoms (r = 0.206; P = 0.015). patients than that found in female patients.25 However, in our study, no significant correlation was found between sex and post-infection antibody levels. The disparities in results may be attributed to sampling and methods used in measuring antibody levels. Furthermore, previous studies showed associ- ations between history of chronic diseases and the levels of antibody.26,27 In our study, no associations were found between history of chronic diseases and the levels of antibody. Besides, in agreement with previous studies,20,28 we found a statistically significant association between the duration of symptoms and antibody levels. This might be explained by that more expo- sure of immune system to the virus with longer duration of symptoms. More research is needed to explore this. Our results are useful because they provide an insight into factors that may impact the production of antibodies in patients with covid-19. Our results can be used by healthcare providers to focus on patients who are at risk of low antibody production. Finally, our results can be used by researchers to investigate antibody production after vaccination in those who are low antibody producers. Declaration of Interests The authors declare no conflict of interest.  study recruiting patients with severe Covid-19, it was shown that the higher levels of antibody was found in female patients than that found in male patients.24 In the same study, the gen- eration of IgG antibody was stronger in females than males in early phase of the disease.24 In contrast, in a study recruiting convalescent patients, antibody levels were higher in male References 1. Kumar A, Singh R, Kaur J, Pandey S, Sharma V, Thakur L, et al. Wuhan to World: The COVID-19 Pandemic. Frontiers in Cellular and Infection Microbiology. 2021;11. 2. Hussein NR. The Role of Self-Responsible Response Versus Lockdown Approach in Controlling COVID-19 Pandemic in Kurdistan Region of Iraq. International Journal of Infection. 2020;7(4). 3. Martinez-Garcia M, Rabasa A, Barber X, Polotskaya K, Roomp K, Oliver N. 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