The Correlation Between IgM and IgG Antibodies With Blood Prole In Patients Infected With COVID 19

Objectives: This study aimed to determine the levels of IgM and IgG antibody response to the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 in coronavirus disease 2019 (COVID-19) patients with different disease severity. Methods: IgM and IgG antibody levels were evaluated via enzyme-linked immunosorbent assay (ELISA). In total, 100 patients with conrmed SARS-CoV-2 infection were enrolled in this study and viral RNA was detected by using Real-time PCR technique. Clinical and laboratory data were collected and analyzed after hospital admission for COVID-19 and two months post-admission. Results: The level of anti-SARS-CoV-2 antibody IgG was signicantly higher in the severe patients than those in moderate and mild groups, 2 months after admission. Also, level of IgG was positively associated with increased WBC, NUT and LYM counts in sever than mild or moderate groups after admission to hospital. Conclusion: Our ndings suggested that patients with severe illness might experience longer virus exposure times and have a stronger antibody response against viral infection. Thus, they have longer time immunity compared with other groups. infection or even resolved after two months [12, 16]. In this study, we assessed the clinical features and the changed levels of IgG and IgM in 100 COVID-19 patients categorized into mild, moderate and sever groups. All patients showed high specic IgG level which suggested they infected with SARS-CoV-2. According to our results, there was a signicant relationship between some clinical symptoms including obesity, fever, and shortness of breath, muscle soreness, odor disorder and taste disorder with the disease severity in COVID-19 patients. However, we found no signicant association between age, sex, blood group, some underlying disease including diabetic, hypertension, cardiovascular disease and asthma disease with disease severity. In contrast with our results, a previous study performed by Sotgiu et al. reported a signicant correlation between age and sex of COVID-19 patients with severity of the illness. They showed that IgM antibody was dramatically increased in patients in the age groups 20–29 years and 60–69 years compared with those aged from 30 to 59 years. Also, they found a statistically signicant higher IgM in males than in females (24.3% VS. 9.1%), showing males were at highest risk of infection and severe disease [33]. Furthermore, our data showed a signicantly association between IgG level and severity of disease. The IgG level was found to be signicantly higher in severe group than mild group, two months after admission. Thus, the sever patients with higher level of IgG had better and longer-term immunity within weeks or months after infection compare to mild and mediated groups. Our results were comparable with several previous ndings of SARS-CoV infections. In compliance with our nding, a previous MERS-CoV study showed that the levels of IgM and IgG antibodies were higher in sever patients compared to patients with mild infection 36]. More studies by Qu and zho et al. reported the delayed IgG and IgM antibody responses as well as higher level of IgG in the critical group compared to non-critical groups 37]. Xie et.al found a higher IgG level in severe than non-sever groups. They also demonstrated a weak correlation between IgM and NEU% percent [37]. In another


Introduction
The novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV)-2, has been identi ed as the causative pathogen of coronavirus disease 2019 (COVID-19) [1][2][3]. On 30 January 2020, World Health Organization (WHO) declared the outbreak of COVID-19 as a public health emergency of international concern [4]. Since December 2019, this serious disease has spread from China to more than 200 countries and territories worldwide via humanto-human transmission ( Fig. 1) [5,6]. The numbers of daily infected cases and COVID-related deaths are still increasing. As of 6 April 2020, a total of Pishtaz Biotechnology Co., Ltd (Tehran, Iran), approved by the Iran Food and Drug Administration (FDA). In this method, the open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) were simultaneously ampli ed and tested by RT-PCR.

SARS-CoV-2 antibody detection
The IgM and IgG antibodies generated against SARS-CoV-2 in serum specimens were detected using IgG and IgM kits supplied by IDEAL TASHKHIS (IDEAL TASHKHIS Co. Tehran, Iran), according to the manufacturer's instructions. The recombinant antigens contain nucleoprotein and spike protein of SARS-CoV-2.
In this IgG and IgM kits 0.9 cut off index. The results ≥ 0.9 were reactive (positive), and the results < 0.9 were nonreactive (negative).

Statistical analysis
The results are presented as mean ± standard deviation (SD) or median and interquartile range. Differences among groups were analyzed using the Mann-Whitney U-test. Statistical analyses were performed using Graph-Pad Prism version 6 (Graph-Pad Software Inc., San Diego, CA, USA). Statistical signi cance was determined to be p < 0.05.

Results
Clinical Symptom and characterize of patients with COVID-19 The present study included a total of 100 hospitalized patients (52(52%) male and 48 (48%) female) with con rmed COVID-19. The patients were classi ed into three clinical groups: mild (22 cases, 22%), moderate (38 cases, 38%) and sever (40 cases, 40%). The average age was 50 years (IQR, 37.25-58.75). The median ages in the severe (51.5 ± 13.5 years) and moderate (52.5 ± 12.5 years) groups were slightly higher than the mild group (43 ± 12.5 years). Thus, there was no signi cant association between patient ages with disease severity. Also, the percentage of males (57.5%) was somewhat more than females (42.5%) in the severe and moderate groups compared to the mild group (males 41%, females 59%) ( Table 1). Moreover, radiological sign of pneumonia was observed at in 78 patients (78%) at the rst clinical evaluation. The most common underlying diseases among the patients were diabetes 37(37%), hypertension 32(32%), cardiovascular disease 15(15%), asthma 14(14%). Some clinical symptoms of patients such as obesity (BMI > 30) (p = 0.05), fever (℃ >39.0) (p = 0.009), shortness of breath (p = 0.015), muscle soreness (p = 0.007), chill (p = 0.025), loss of consciousness (p = 0.041), odor disorder (p = 0.036), anorexia (p = 0.029) and taste disorder (p = 0.027) were signi cantly associated with severity of disease. However, we nd no signi cant correlation between age, sex, blood group and the underlying disease such as diabetic, hypertension, cardiovascular disease, organ failure, asthma disease, also other symptoms such as fatigue, sore throat, chest pain, headache, nausea, vomiting, diarrhea, visual impairment in patients with severity of disease (Table 1). Several laboratory markers such as ferritin, LDH and D-dimer were increased, while brinogen level was decreased in most patients at baseline after admission in hospital (step1) compare to two months after admission for COVID-19 (step2). Liver injury marker such as AST, ALT and ALP were also signi cantly increased at baseline ( Table 2). In addition, we found that the higher WBC, NUT counts as well as lower LYM counts were correlated with the increased level of IgG two months after admission, resulting a longer time of immunity. It follows that the patients in sever group has better and longer immunity compare to mild groups (Table 3).  Interestingly, we found some abnormalities laboratory ndings in the sever groups and non-severe groups. Our data showed that the IgG levels in severe group was signi cantly higher than mild groups. This also included higher WBC, higher neutrophil and lower lymphocyte counts among the groups (p < .05) (Fig. 2).

Discussion
The COVID-19 pandemic has focused attention to crucial role of diagnostic techniques in controlling infectious diseases. The standard current diagnostic methods used for SARS-CoV-2 infection based on the stage of disease are nucleic acid-based molecular tests (RT-PCR) and antibody-based tests (Serologic tests). The RT-PCR is used for the early detection of the infection and target SARS-CoV-2 N gene and ORF1ab, while serological test is applied for assessing the disease progression [31,32]. After SARS infection, IgM antibodies are produced by immune cells during the early stages of infection, followed by IgG generated in the later stages of SARS-CoV-2 infection. The detection of IgM antibody indicates a recent exposure to SARS-CoV-2 and the detection of IgG antibody in the absence of detectable IgM antibody, indicates prior virus exposure [13,16]. IgG immunoglobulins are monomeric antibodies in the serum and crucial in maintaining long-term immunity or immunological memory after infection [33]. In general, IgM is detectable after 3-6 days, and IgG is detectable after 8 days [34], while viral RNA may be undetectable even after two weeks due to its rapidly decreased level [12]. Therefore, the IgM and IgG antibodies become the main and most accurate procedure to detect an active SARS-CoV-2 infection or even resolved after two months [12,16]. In this study, we assessed the clinical features and the changed levels of IgG and IgM in 100 COVID-19 patients categorized into mild, moderate and sever groups. All patients showed high speci c IgG level which suggested they infected with SARS-CoV-2. According to our results, there was a signi cant relationship between some clinical symptoms including obesity, fever, and shortness of breath, muscle soreness, odor disorder and taste disorder with the disease severity in COVID-19 patients.
However, we found no signi cant association between age, sex, blood group, some underlying disease including diabetic, hypertension, cardiovascular disease and asthma disease with disease severity. In contrast with our results, a previous study performed by Sotgiu et al. reported a signi cant correlation between age and sex of COVID-19 patients with severity of the illness. They showed that IgM antibody was dramatically increased in patients in the age groups 20-29 years and 60-69 years compared with those aged from 30 to 59 years. Also, they found a statistically signi cant higher IgM in males than in females (24.3% VS. 9.1%), showing males were at highest risk of infection and severe disease [33]. Furthermore, our data showed a signi cantly association between IgG level and severity of disease. The IgG level was found to be signi cantly higher in severe group than mild group, two months after admission. Thus, the sever patients with higher level of IgG had better and longer-term immunity within weeks or months after infection compare to mild and mediated groups. Our results were comparable with several previous ndings of SARS-CoV infections. In compliance with our nding, a previous MERS-CoV study showed that the levels of IgM and IgG antibodies were higher in sever patients compared to patients with mild infection [35,36]. More studies by Qu and zho et al. reported the delayed IgG and IgM antibody responses as well as higher level of IgG in the critical group compared to non-critical groups [17,37]. Xie et.al found a higher IgG level in severe than non-sever groups. They also demonstrated a weak correlation between IgM and NEU% percent [37]. In another study done by Park et al. on ESH, ZA and HHK were responsible for the study conception and design and HB, HE, SS, ARO, MH. MS and NRK performed data collection and HSH, ZA and HHK preparing the rst draft of the manuscript. MJA did the data analysis, HHK made critical revisions to the paper for important intellectual content and supervised the study.