key: cord-0986674-oihyz5v7
authors: Zhang, Guoxin; Nie, Shuke; Zhang, Zhaohui; Zhang, Zhentao
title: Longitudinal Change of SARS-Cov2 Antibodies in Patients with COVID-19
date: 2020-05-02
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa229
sha: acf315f592d4df412224954a629679da3a30372f
doc_id: 986674
cord_uid: oihyz5v7

BACKGROUND: A novel coronavirus, SARS-CoV-2, has recently emerged and caused the rapid spread of COVID-19 worldwide. METHODS: We did a retrospective study and included COVID-19 patients admitted to Renmin Hospital of Wuhan University between February 1 and February 29, 2020. Antibody assay was conducted to detect COVID-19 envelope protein E and nucleocapsid protein N antigen. RESULTS: 112 patients were recruited with symptoms of fever, cough, fatigue, myalgia, and diarrhoea. All patients underwent antibody tests. Fifty-eight (51.79%) were positive for both IgM and IgG, 7 (6.25%) were negative for both antibodies, 1 (0.89%) was positive for only IgM, and 46 (41.07%) were positive for only IgG. IgM antibody appeared within a week post disease onset, and lasted for one month and gradually decreased, while IgG antibody was produced 10 days after infection, and lasted for a longer time. However, no significant difference in level of IgM and IgG antibody between positive and negative patients of nucleic acid test after treatment was found. CONCLUSIONS: Our results indicate that serological tests could be powerful approach for the early diagnosis of COVID-19.

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In December 2019, a rapidly spreading coronavirus disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred in Wuhan, China [1] . Currently, the disease has emerged as an explosive epidemic in many countries, showing the characteristics of a global pandemic. Whole-genome sequencing results show that COVID-19 is classified under the betacoronavirus 2B family because of its typical coronavirus family characteristics [2] , of which the severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) are well known to people because of their previous outbursts. The genome sequence data of SARS-CoV-2 showed more than 80% identity with SARS-CoV and 50% identity with MERS-CoV [3, 4] . While the origin of SARS-CoV-2 remains unclear, current evidence suggests its transmission from bat to humans through a potential intermediate host [5] . Autopsy results show that inflammatory storms play an important role in the pathological changes of the disease. The latest reports show that CD4+ and CD8+ T cell counts in the peripheral blood of SARS-CoV-2-infected patients are significantly reduced [6] . The most common clinical manifestations of COVID-19 include fever, dry cough, and fatigue. Other symptoms include sputum production, myalgia, headache, and diarrhoea [7, 8] .

Currently, there is still no effective drug for COVID-19 treatment, and the vaccine is in the stage of clinical trials. Early diagnosis, isolation, and treatment are essential to cure the disease and control the epidemic. Serum antibody detection is of great significance in the diagnosis of infected patients, especially for patients with negative nucleic acid test. Simultaneous detection of both IgM and IgG antibodies helps to identify the stage of the infectious. Generally, the antibody profile against SARS-CoV has a typical pattern of IgM and IgG production. The SARS-specific IgM antibodies appeared in about two weeks after infection, and disappeared at the end of week 12, while the IgG antibodies last for months or even many years [9] . For COVID-19, however, the longitudinal pattern of the antibodies remains unclear. We performed this study to investigate the potential relationships between immune antibodies and disease progression.

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Epidemiological, clinical, and laboratory information was obtained with data collection forms from electronic medical records and were reviewed by trained physicians. The date of disease onset was defined as the day when the symptom was noticed. Nasopharynx swab and oropharynx swab samples were collected and tested for SARS-CoV-2 open reading frame 1ab(ORF1ab)/nucleocapsid protein (N) gene with the double nucleic acid detection kit (BioGerm, Shanghai, China), following WHO guidelines [10, 11] . IgM and IgG antibody detection kit (Yahuilong Biotechnology, Shenzhen, China) was developed to detect COVID-19 envelope protein E and nucleocapsid protein N antigen. The background antibody titre in uninfected healthy individuals is under 10 AU/ml. Any test that is higher than 10 AU/ml was considered as positive.

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Statistical analysis was performed using SPSS, version 20.0. Continuous variables were expressed directly as a range. Categorical variables were expressed as numbers (%). Two-sided P < 0.05 was considered statistically significant.

This retrospective study included 112 patients, with 33 (29.5%) males and 79 (70.5%) females.

All patients had a positive result in the nucleic acid test. The median age of the subjects was 38.625 ± 14.9 years (25-78 years). With the exception of 10 patients who reported no symptoms (8.93%), most common symptoms were fever (61, 54.46%), cough (52, 46.43%), fatigue (29, 25.89%), dizziness (2, 1.79%), pharyngeal pain (15, 13.39%), diarrhoea (11, 9 .82%), vomiting (2, 1.79%), myalgia (2, 1.79%), headache (4, 3.57%), and eye discomfort (1, 0.89%) ( Table 1) . Most of the participants in this study were young people without previous medical history. All patients had mild symptoms, and no one was sent to the intensive care unit. Of the 11 patients with diarrhoea, 3 (27.27%) were anal swab positive.

Serological antibody tests were performed at different times post disease onset. The overall antibody positivity was 93.75% (105/112). Fifty-eight (51.79%) of 112 patients were positive for IgM (20.93 ± 45.94 AU/ml, mean ± SD) and IgG (122.26 ± 60.94 AU/ml), 7 (6.25%) were negative for both antibodies, 1 (0.89%) was positive for IgM with no response to IgG, and 46 (41.07%) were positive for IgG but not for IgM. Further group subtypes were analysed based on the course of the disease ( Table 2 ). Compared to the IgG titres tested within 10 days after the onset of COVID-19, the IgG titers tested 20-30 days (p=0.0025), 30-40 days (p=0.0147) and 40-50 days (p=0.0049) after disease onset were significantly higher ( Table 2 ). Figure 1 shows the distribution of antibody according to the time points after disease onset.

A c c e p t e d M a n u s c r i p t 7 Among the 7 patients who were tested for serological antibody within 10 days after the onset of the disease, 4 were positive for both antibodies (6-8 days after disease onset), one was positive for only IgM (4 days after the onset of the disease), and two patients were negative for both antibodies.

Among the 10 patients who underwent serological antibody testing 10-20 days after disease onset, 5 were positive for both IgM (37.42±18.69) and IgG (161.19±16.80) antibodies, 3 were positive for IgG (43.46±20.42). Two subjects were negative for both IgM and IgG antibodies. Furthermore, only the initial PCR test was positive for these two subjects. All the subsequent PCR test were negative. Of the 112 patients included in this study, 26 underwent two successive antibody and nucleic acid tests. Eleven were positive for the second nucleic acid test, and 15 were negative. Of these 26 patients, the initial IgM and IgG titters were 21.65 ± 26.35 AU/ml and 112.96 ± 47.67 AU/ml, respectively. The positivity rates of IgM and IgG were 50% (13 patients) and 100% (26 patients), respectively. Of the 11 patients who were positive for the second nucleic acid test, the initial IgM and IgG titters were 22.83 ± 35.39 AU/ml and 106.78 ± 44.31 AU/ml, respectively. The positivity rates of IgM and IgG were 45% (5 patients) and 100% (11 patients), respectively. Of the 15 patients who were M a n u s c r i p t 8 negative for the second nucleic acid test, the initial IgM and IgG titters were 20.79 ± 18.55 AU/ml and 112.59 ± 51.03 AU/ml, respectively. The positivity rates of IgM and IgG were 87% (13 patients) and 100% (15 patients), respectively. (Table 3 ). In addition, Figure 2 showed the trend lines for the IgM and IgG antibody levels at visit one and two.

In addition, we made sub-groups analysis according to the age of the patients ( 

The COVID-19 diagnosis and treatment plan recommended RT-PCR for the detection of SARS-CoV-2 nucleic acid. A positive nucleic acid test is needed for the diagnosis of suspected cases.

However, the diagnostic value of nucleic acid detection is greatly affected by the sample quality, experimental conditions, and operation protocols. Serological surveys can aid the investigation of an ongoing outbreak and retrospective assessment of the attack rate or extent of an outbreak. In cases where there is a strong epidemiological link to COVID-19 infection, paired serum samples (in the acute and convalescent phase) is another key evidence for the diagnosis of infection. The antibody detection is simple and repeatable, with a low risk of infection for medical staff during the process of sample collection and detection, and thus is an important way of rapid screening.

Among the 112 patients included in this retrospective study, 1 patient presented with dry cough was positive for IgM and negative for IgG. This patient's IgM antibodies appeared in about 4 days post disease onset, the time of which was earlier than SARS-specific IgM antibodies that appeared in about two weeks after infection, and disappeared at the end of week 12 [9] . This finding highlights the A c c e p t e d M a n u s c r i p t 9 importance of collecting the serum sample of COVID-19 patients as early as possible. However, the titre of IgM antibody is usually low, and lasts for a short time. Contrarily, IgG production indicates the middle and later stages of infection or previous infection, along with high concentration, longer duration, and higher affinity. When IgG in a patient during convalescence is 4 times or higher than that in the acute period, it indicates recurrent infection. Among the 7 patients who were tested for serological antibody within 10 days after onset of the illness, 2 were negative for IgM antibody. These subjects might be in the 'window period' of SARS-CoV-2 infection. During this period, it is difficult to detect the antibody. The advantage of nucleic acid detection over serum antibody detection is that it shortens the detection window of infection.

Two subjects were negative for both IgM and IgG 10-20 days after disease onset. They were negative for all subsequent PCR tests performed after the initial positive test. Considering the quickly relieved symptoms of headache and sore throat, the result of a nucleic acid test in the first time might be false positive.

Time of antibody responses begin to appear over a period of days to weeks after SARS-CoV-2 infection, to some extent of which are dependent on the sensitivity of the detection and the viral protein used as an antigen. In our study, patients were detected positive for IgM by enzyme immunoassay to nucleoprotein as early as 4 days, the time of which was consistent with the observation shown that IgM can be detected 3-6 (median 5) days after onset of CoVID-19 symptoms [12, 13] . However, IgG in our design was seen 6-8 days after disease onset, earlier than that is detected 10-18 (median 14) days after onset of symptoms [12] . Tan et al. reported that the antinucleocapsid-protein IgM started on day 7 and positive rate peaked on day 28, while that of IgG was on day 10 and day 49 after disease onset [14] . However, antibody to the receptor-binding domain of the spike protein was detected a median of 11 days after onset of symptoms, but the timing of seroconversion did not correlate with clinical course [13, 15] . Moreover, studies in patients with SARS and MERS suggest that antibody responses for SARS-CoV-1 and MERS-CoV are not durable [16, 17] A c c e p t e d M a n u s c r i p t 10 samples tested 6 years after SARS-CoV-1 infection [19] . It is still unclear whether the antibodies to SARS-CoV-2 also disappear within years.

In addition, studies in subjects infected with MERS-CoV found that antibody levels were higher in those experienced severe infection compared to those with mild infection [17] . With regard to SARS-CoV-2, titres of IgM and IgG are significantly higher in severe patients than non-severe patients (p<0.05). The weak responders for IgG had a significantly higher viral clearance rate than that of strong responders [14] . Similar result was found in another study reporting a higher titre of antibody was independently associated with a worse clinical classification [13] . However, only patients with mild symptoms were included in our study, and there was no difference between nucleic acid positive and negative patients when retesting the antibody titres. Owing to the small sample size, whether the elderly patients are more likely to cause the increase of antibody titre still needs to be further tested.

There are many limitations in this study. First, the antibody detection kit was designed for COVID-19 envelope protein (E) and nucleocapsid protein (N) antigen but not for the Spike protein (S). S protein is believed to be the key site that mediates the human-to-human transmission by binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2) [5] . Second, limited to this singlecentre study, it is necessary to investigate the relationship between the dynamic change of antibody and the course of COVID-19 in a multicentre study with a larger sample size. Third, the longitudinal changes of antibodies should be traced to understand the disease progression.

In summary, in addition to being used for the supplementary detection of SARS-CoV-2 nucleic acid test negative cases, antibody detection can, to some extent, track disease progression. Through retrospective analysis, we found that antibody test is useful for the early diagnosis of COVID-19. IgM antibody appeared within one week after SARS-CoV-2 infection, and this antibody was present in the body for one month or even longer and then A c c e p t e d M a n u s c r i p t 11 gradually decreased until it was lower than the detection limit. IgG antibody is usually produced in about 10 days, but the time it will persist in body remains unclear. However, after treatment, no significant difference in the level of IgM and IgG antibodies was found between nucleic acid-positive and negative patients. Further investigation of duration of protective immunity for SARS-CoV-2 and acquired immunity to reinfection will be critical to M a n u s c r i p t 17 

Outbreak of pneumonia of unknown etiology in Wuhan, China: The mystery and the miracle

The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health -The latest 2019 novel coronavirus outbreak in Wuhan, China

Identification of a novel coronavirus causing severe pneumonia in human: a descriptive study

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

Receptor recognition by novel coronavirus from Wuhan: An analysis based on decade-long structural studies of SARS

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

Updated understanding of the outbreak of 2019 novel coronavirus (2019-nCoV) in Wuhan

Profile of specific antibodies to the SARS-associated coronavirus

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

COVID-19 ) in suspected human cases. Interim guidance

Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19)

Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease

Viral Kinetics and Antibody Responses in Patients with

Virological assessment of hospitalized patients with COVID-2019

Duration of antibody responses after severe acute respiratory syndrome

Antibody Response and Disease Severity in Healthcare Worker MERS Survivors

Two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome

Lack of peripheral memory B cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study

The authors declare no conflict of interests.

This work was supported by grants from National Natural Science Foundation of China (No.81822016 and 81771382) to Z. Zhang.

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