SARS-CoV-2 Antibody Responses in Newly Recovered Patients from Covid-19 Infection

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Introduction
The coronavirus disease 2019 (COVID-19) pandemic continues, causing considerable morbidity and mortality worldwide.The severity of COVID-19 ranges from asymptomatic to fatal pneumonitis, with mildly symptomatic patients accounting for approximately 80% of all cases according to current understanding [1].Full-genome sequencing and phylogenic analysis indicated that SARS-CoV-2 belongs to the betacoronavirus 2b lineage, the same group as Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), another highly virulent pathogens in humans.Bats are suspected to be the reservoir and pangolins are suggested to be an intermediate host for SARS-CoV-2 [2,3].Initial clinical manifestations of the disease include fever, fatigue, and dry cough, while patients with severe disease may exhibit pneumonia and acute respiratory distress syndrome (ARDS) [4].
Apart from supportive care, specific drugs for this disease are still being researched [5,6].Real-time reverse trans cription polymerase chain reaction (RT-PCR) diagnostic assays are a goal standard for case ascertainment and diagnosis [7].
Many cases that were strongly epidemiologically linked to SARS-CoV-2 exposure and with typical lung radiological findings remained RNA negative in their upper respiratory tract samples.The performance of RT-PCR depends on many factors such as the sample types [8], different stage of infection in patients [9] the skill of sample collection, and the quality and consistency of the PCR assays being used.A nasopharyngeal specimen is the preferred choice for swab-based SARS-CoV-2 testing, but oropharyngeal sample should also be collected whenever feasible [9,10].
These problems lead to a noteworthy delay of early diagnosis and following management and propose serious challenge to providing timely life support treatment and preventive quarantine.Comparing to PCR, serological testing is advantageous with faster turnaround time, high-throughput and less workload.However, the clinical value of antibodies largely depends on the understanding of host antibody responses during infection.Given that SARS-CoV-2 is a newly emerging virus, the antibody response in COVID-19 patients remains largely unknown [11].
The aim of this study is To investigate the dynamics of the (Immunoglobulin M (IgM) and Immunoglobulin G (IgG)) in response to SARS-CoV-2 in patients newly recovered from different severity Covid-19 infection.

Patients and Methods
This cross sectional study where done in the Central blood bank of Diyala for the period from 1st June 2020-1st August 2020.(93) newly recovered patients with documented SARS-CoV-2 infection by using reverse transcriptase polymerase chain reaction test (RT-PCR) who came for convalescent plasma donation were enrolled in this study.The questionnaire form was filled including the patient name, age, address, gender, clinical manifestations at time of the infection, the severity and the RT-PCR results.

VIDAS SARS-CoV-2 IgM and VIDAS SARS-CoV-2 IgG test:
This test done by taking (5ml) blood from the patient, then centrifuged to separate the serum.Then we take a small amount of the serum and test for IgG and IgM antibodies by using combines a two-step sandwich enzyme immunoassay method with a final fluorescence detection.This test done by using Minividas machine made by France at 2016 which is available in the central blood bank-Diyala.

Statistical Analysis
Statistics is an advanced analytical software and solutions package originally developed by StatSoft.The program includes a combination of data analysis, data management, statistics, machine learning, text analytics and data visualization procedures, and data mining procedures; In addition to a variety of predictive modeling, clustering, classification, and exploratory techniques.

Results
Male constitute about (91%) of the study group.Only 26% cases while 74% cases below (40) years old.(54%) of the patients had mild disease, (28%) had moderate severity while the other (18%) had severe type of the disease.About comorbidities (6%) of the cases had chronic bronchitis, (4%) of the cases had diabetes mellitus, (3%) had hypertension and (1%) had chronic renal disease.The demographic characteristics of them were shown in Table (1).The most common symptoms of the patients under the study during the time of infection were fatigue and fever and seen in (87% and 84%) respectively, while asymptomatic patients were seen in (2%) only.As shown in Table (2).Although mild cases show early seroconversion (before 21 day) but only (67%) had IgM Antibodies after (49) day post infection, while severe type show late appearance of IgM Antibodies (after 21 day) and at (49) day post infection all patients with severe type of the disease (100%) were IgM positive.
IgG Antibodies were positive in (100%, 0%) of the mild and severe cases respectively at (21) day post infection but it's positive in (100%) of both mild and severe cases after (49) day post infection.This indicate that although the IgG antibodies appear slowly and later than IgM antibodies but it last longer in the circulation mainly in the severe type of SARS-CoV-2 infection.

Discussion
In this study the male patients constitute (91%) of the cases and this agree with the study done in Iraq in 2021 [12], another study done in Iraq in 2021 [13],a study done in Bangladesh in 2020 [14] and a study done in Sweden in 2020 [15] which show that male constitute about (67%,63%, 69% and 62%) respectively.While in other studies like a study from China 2020 [16] and a study from Thailand in 2020 [17] which show that male constitute only (39.5% and 40%) respectively.And this may be due to the circumstances under which the study were done.Also many factors, including innate immunity, regulatory T cells, expression of angiotensin-converting enzyme 2 (ACE2), or other mechanisms related to sex hormones might explain the difference in immunity between both sex.
The median age group affected in this study were (34) and the most affected age group was (20-29).This is lower than the finding in many studies like that from Sweden in 2020 [15],a study from China in 2020 [16] and a study from Thailand in 2020 [17] which show a median age were(45, 47 and 38% respectively)and this may be due to the fact that this is the age of most workers and others who participate in social activities and easily get the infection there.
The main chronic disease in our study patients were chronic bronchitis which is found in about (6%).While in other studies like that from Sweden in 2020 [15] and from China in 2020 [16] it was hypertension in (9% and 23% respectively).This may be due to the prevalence of these chronic illnesses in our community.So many patients were smokers and had chronic bronchitis which put them at risk for Covid-19 infection.
The main symptoms were fatigue and fever and seen in (87% and 84%) respectively, this agree with a previous study done in Iraq in 2021 [12] and a study done in Bangladesh in 2020 [14] which show the main symptom was fever and seen in (79.5% and 76%) respectively.
The IgM Antibodies starts to appear in the circulation before (21) days post infection mainly in mild and moderate types (100%) and in the severe cases (0%) were positive at this period and this agree to some degree with the study done in China in 2020 [18] which show that the positive rate for IgM in the mild group was 100%, compared to 75% in the severe group at (15-21) day post infection.And these findings go with the suggestion that the appearance of noticeable levels of SARS-CoV-2 IgM antibodies in severe cases of COVID-19 is delayed.
While after (49) day from the time of the infection (67%and 75%) of the mild and The IgG Antibodies were found to be positive in (100%,0%) of the mild and severe cases respectively at (21) day post infection but it's positive in (100%) of both mild and severe cases after (49) day post infection.This disagree with the study done in Thailand in 2020 [17] which show that only (50%) of the mild cases and (100%) of both moderate and severe cases were positive at day (15-28) post infection.But it goes with the findings in other studies like that from Sweden in 2021 [15]which show that clinical severity of the disease is associated with higher SARS-CoV-2-specific serum-IgG antibodies that although it appears late in the course of the disease but it lasts longer and further studies with long follow up period needed to prove it.

Conclusions
Although the serological response for SARS-CoV-2 appears in the first three weeks from the disease onset this response differs from patient to another according to the time from the infection and the severity of the infection.

Recommendations
Further studies were needed to follow up post SARS-CoV-2 patients for longer periods to assess the exact duration of these antibodies

Source of funding:
The current study was funded by our charges with no any other funding sources elsewhere.
Volume 25, Issue 2 moderate cases respectively where IgM positive and (100%) of the severe cases were IgM positive.So, this suggest that the IgM Antibodies appear early and decline early in mild-moderate cases while in severe cases it raises slowly and decline later in the course of the illness.

Table ( 1
): : The demographic characteristics of the study group

Table ( 2
): The main symptoms of the patients at time of infection