key: cord-1025718-q88q4tnb
authors: Bai, Zhenjiang; Li, Qing; Chen, Qinghui; Niu, Changming; Wei, Yu; Huang, Hanpeng; Zhao, Wei; Chen, Nian; Yao, Xin; Zhang, Qiang; Mu, Chuanyong; Feng, Jian; Zhu, Chuanlong; Li, Zhuo; Ding, Ming; Feng, Binhui; Jin, Chaochao; Lu, Xiang; Yang, Yi; Wu, Shuiyan; Shu, Xiaochen; Hu, Lifang; Qiu, Haibo; Huang, YingZi
title: Clinical Significance of Serum IgM and IgG levels in COVID-19 patients in Hubei Province, China
date: 2021-10-04
journal: Journal of Intensive Medicine
DOI: 10.1016/j.jointm.2021.09.001
sha: 7434e54b03d24cce2e859f8b45f8645a51432385
doc_id: 1025718
cord_uid: q88q4tnb

Background There have been many studies about coronavirus disease 2019 (COVID -19), but the clinical significance of quantitative serum severe acute respiratory syndrome-coronavirus-2(SARS-CoV-2)-specific IgM and IgG levels in COVID-19 patients have not been exhaustively studied. We aimed to study the time profiles of these IgM/IgG levels in COVID-19 patients and their correlations with clinical features. Methods A multicenter clinical study was conducted from February to March 2020. It involved 179 COVID-19 patients (108 males and 71 females) from five hospitals in Huangshi in Hubei Province, China. To detect SARS-CoV-2-specific IgM/IgG, quantitative antibody assays (two-step indirect immunoassays with direct chemiluminescence technology) based on the nucleocapsid protein (NP) and spike protein 1 (S1) were used. For normally distributed data, means were compared using the t test, χ2 test, or exact probability method. For categorical data, medians were compared using Mann-Whitney U test. Results The median age was 57(44-69) years (58(38-69) for males and 57(49-68) for females). The median duration of positive nucleic acid test was 22.32 (17.34-27.43) days. The mortality rate was relatively low (3/179, 1.68%). Serum SARS-CoV-2-specific IgG was detected around week 1 after illness onset, gradually increased until peaking in weeks 4 and 5, and then declined. Serum IgM peaked in weeks 2 and 3, then gradually declined and returned to its normal range by week 7 in all patients. Notably, children had milder respiratory symptoms with lower SARS-CoV-2-specific IgM/IgG levels. The duration of positive nucleic acid test in the chronic obstructive pulmonary disease (COPD) group was 30.36(18.99-34.76) days, which was significantly longer than that in the non-COPD group (21.52(16.75-26.51) days; P=0.011). The peak serum SARS-CoV-2-specific IgG was significantly positively correlated with the duration of positive nucleic acid test. The incidence rate of severe and critical cases in the IgMhi group (using the median IgM level of 29.95 AU/ml as the cutoff for grouping) was about 38% (19/50), which was twice as much as that in the IgMlo group (18.37%; 9/49). The patients with positive chest imaging and lymphocytopenia (<1 × 109/L) had a higher SARS-CoV-2-specific IgM level. Conclusions Quantitative SARS-CoV-2-specific IgM and IgG levels are helpful for the diagnosis, severity classification, and management of COVID-19 patients, and they should be monitored in each stage of this disease.

The coronavirus disease 2019 (COVID-19) outbreak, which was first observed in Wuhan, China, and reported to the World Health Organization (WHO) in December 2019, was characterized as a pandemic by the WHO on March 11, 2020 (1) . Severe acute respiratory syndrome-coronavirus-2(SARS-CoV-2 ) is a completely novel threat to human health (2) . There are still many questions about COVID-19 that need to be urgently answered. For example, unpredicted sudden deterioration and death can 6 occur in patients who were formerly in a stable condition, and some convalescent patients who had two or more negative nucleic acid tests subsequently exhibited positive results. There is currently no reasonable explanation for these phenomena from the immunological point of view, and it is urgent that we improve our understanding of the human immune response in each stage of the disease.

Detection of virus-specific antibody is effective for the diagnosis of SARS-CoV and middle east respiratory syndrome-coronavirus (MERS-CoV ) infection (3) (4) (5) .

Currently, according to "Diagnosis and Treatment Plan of Coronavirus Disease 2019 (Tentative 7th Revised Edition)," SARS-CoV-2 antibody assays are extensively carried out for COVID-19 diagnosis (3). Using bat SARSr-CoV Rp3 nucleocapsid protein (NP) as the antigen, Peng et al. (6) identified dynamic changes in antibodies in the peripheral blood of COVID-19 patients. However, several studies raised concern over the method accuracy, as research revealed that using intact NP as the antigen for serological detection may affect the assay specificity (3, 7) . Here, we used quantitative antibody assays based on the NP antigen and the spike protein 1 (S1) antigen in order to gain a better understanding of the human humoral immune response to SARS-CoV-2 infection. This may improve future clinical diagnosis, severity classification, and management of COVID-19. We present this article in accordance with the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) checklist. 7 This study was a multicenter descriptive study with a duration of 7 weeks. It was conducted in compliance with the principles of the Declaration of Helsinki and approved by the Clinical Research Ethical Committee of Zhongda Hospital, Affiliated with Southeast University (approval number: 2020ZDSYLL018-P01). Written informed consent was provided by the patients or their family members.

The study included 179 patients who were admitted to Huangshi Central Hospital, 

Peripheral blood samples (2 ml) were collected from each patient, weekly from the day of admission to discharge, using a serum separator transport tube (SST; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). SST tubes contain a polymer separation gel that separates the cellular clotted material from the serum, so the serum is located above the polymer barrier after centrifugation. The upper layer (serum) was 8 used for further analysis.

We used quantitative antibody assays based on the NP plus spike protein 1 (S1) antigens in order to gain a better understanding of the human humoral immune response to SARS-CoV-2 infection. A two-step indirect immunoassay with direct chemiluminescence technology (Shenzhen Yahuilong Biotechnology Co., Ltd., China) was used, according to the kit specification. The cutoff was 10 AU/ml, so the result was deemed to be non-reactive if the antibody level was <10.00 AU/ml. To ensure appropriate test performance, the quality control products were tested every 24 hours or after each calibration.

Upper airway specimens (pharyngeal swabs and nasopharyngeal secretions) and lower airway specimens (sputum and airway secretions) were obtained at admission according to standard procedures. All laboratory procedures were carried out in a Biosafety Level 3 laboratory, according to the SARS-CoV-2 nucleic acid test kit instructions (BGI Biotechnology, Co., Ltd., Shenzhen, China). Total RNA was extracted using a nucleic acid extraction kit (DP315R; TIANGEN, Beijing, China). Thereafter, 10 µL RNA was used for real-time quantitative PCR, which was performed under the following conditions: 50°C for 20 min, 95°C for 10 min, and 40 cycles of amplification at 95°C for 15 s and 60°C for 30 s. The cutoff value of the kit was determined using a receiver operating characteristic curve(Ct≤38) 9 The diagnosis and severity of COVID-19 were defined according to "Diagnosis and Treatment Plan of Coronavirus Disease 2019 (Tentative 7th Revised Edition)." Lymphocytopenia (peripheral blood lymphocyte count <1×10 9 /L) was determined based on the peak value detected in week 1.

Data were analyzed using SPSS 22.0 software (IBM Corporation, Chicago, Illinois, USA). Normally distributed continuous data are expressed as mean ± standard deviation. The mean was compared between pairs of groups using the t test, χ 2 test, or exact probability method. Categorical data are expressed by median and interquartile range (IQR). The median was compared between pairs of groups using the Mann-Whitney U test. Two-tailed P<0.05 was considered statistically significant. Table 1 .

The percentages of cases with serum IgG detection from week 1 to 7 after illness onset were 50.00% (3/6), 100.00% (24/24), 100.00% (69/69), 100.00% (60/60), 100.00% (25/25), 100.00% (12/12), and 100.00% (6/6), respectively, while the percentages with serum IgM were 50.00% (3/6), 75.00% (18/24) 

All children (<18 years old; n=9) had mild or moderate clinical symptoms. The median age of children was 6 (2.34-13.5) years, and that of adults (n=170) was 58 (48-69) years. The median IgG level from week 2 to 5 in children was 73.08(2.29-151.73) AU/ml, which was significantly lower than that in adults 146. 28 Table 2 ). 11 The percentage of patients with underlying diseases (COPD, diabetes, hypertension, coronary heart disease, and/or cerebral infarction) in the critical group was 60.71% (17/28), which was significantly higher than that in the mild group (12.00%; 3/25; P=0.007) and moderate group (37.00%; 37/100; P=0.025).

The duration of positive nucleic acid test in the COPD group (n=14) was 30 .36(18.99-34.76) days, which was significantly longer than in the non-COPD group Table 2 ). There was no significant difference in the serum SARS-CoV-2-specific IgM/IgG ratio between the COPD and non-COPD groups (P>0.05). There were also no significant differences in the duration of positive nucleic acid test and SARS-CoV-2-specific IgM/IgG ratios between groups with or without underlying diseases (P>0.05).

The mean duration of positive nucleic acid test was 21. 

The incidence rate of lymphocytopenia in hospitalized COVID-19 patients was 33.52% (60/179). The median serum SARS-CoV-2-specific IgM level was 35.24(17.61-103.44) AU/ml in the lymphocytopenia group (n=60), which was significantly higher than that (22.83(9.89-63.59) AU/ml) in the non-lymphocytopenia group (n=119; P=0.038; Table 2 ).

The Table 2 ). The serum SARS-CoV-2-specific IgG level in the oxygen therapy group was 144.74(100.56-165.94) AU/ml, which was significantly higher than that in the non-oxygen therapy group (120.97(78.64-162.46) AU/ml; P=0.045; Table 2 ).

The duration of positive nucleic acid test in mild cases was 17.89(13.42-24.60) days, which was significantly shorter than that in non-mild cases (22.52(17.49-27.47) days; P=0.022; Table 3 ).

It is well-known that the production of antibodies against a particular virus is very similar across patients (except for immunocompromised patients) during the acute phase of infection. Although SARS-CoV NP has high sensitivity for SARS, its specificity is not sufficient, as it cross-reacts with antibodies against several class I animal coronaviruses such as human coronavirus 229E, feline infectious virus, and pig infectious virus (7, 8) . Therefore, using intact NP as an antigen for serological detection regarding SARS-CoV-2 may reduce the assay specificity. The S protein of SARS-CoV-2 is composed of the S1 and S2 subunits (9, 10). The S1 subunit contains the receptor binding domain (RBD), which is responsible for binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2). The S2 subunit mediates the subsequent membrane fusion process (9, 11) . We assessed antibodies targeting the NP and S1 proteins of SARS-CoV-2 instead of NP alone in order to enhance the specificity of the assay.

Our results showed that serum SARS-CoV-2-specific IgG was detected around week 1 after illness onset, gradually increased until peaking in weeks 4 and 5, and then declined but was still maintained at a higher level than that in week 1. Serum IgM appeared around week 1 and peaked in weeks 2 and 3, earlier than serum IgG did.

It then gradually declined and returned to the normal range by week 7 in all patients.

Although the trend was similar, the details such as the peak timing and the duration of the antibodies differed from those in SARS and MERS. Lee et al. found that serum SARS-CoV IgG was first detected on day 4 after illness onset, seroconversion 14 occurred at a median of 16 days (range: 4 to 35 days), and IgG peaked in week 4 (4).

Another study revealed that serum SARS-CoV IgG increased after week 1 and peaked on day 60, and remained at a high level until month 6, at which point it declined gradually until month 24. IgM was detected around day 15 and rapidly peaked, and then declined until it was undetectable after 6 months (12). MERS-CoV IgG was still detectable in patients with MERS infection in month 12 (5) . In contrast, in our study, the IgM level in all patients dropped back to the normal range by week 7. We did not have data on the persistence of detectable SARS-CoV-2-specific IgG; however, 100% of patients had increased IgG level at week 7.

Regarding age, we found that children have milder respiratory symptoms, a shorter duration of positive nucleic acid test, and lower SARS-CoV-2-specific IgM/IgG levels. During the SARS-CoV pandemic, most children had a benign course of illness with milder symptoms and there were no deaths reported for children (13) .

However, comparing the data from children and adults with SARS, Previous study found no significant differences in plasma viral load within week 1 after admission (14) . One explanation is a relatively blunted immune response against SARS-CoV in children (15) (16) (17) . Another explanation is the increased presence of high-affinity IgG against the common circulating human coronavirus strains in adults. Children aged <6 years lack anti-CoV IgG, and then they start to develop antibodies against the common circulating coronavirus strains in humans (NL63, 229E, OC43, and HKU1).

The anti-CoV repertoire in children consists of IgG with low affinity, which will gradually mature into high-affinity anti-CoV IgG after repeated infections (14, 18) . 15 We found that patients with COPD had a longer duration of positive nucleic acid test. Kwak et al. reported that patients with COPD had worse outcomes during viral infections such as rhinovirus, respiratory syncytial virus, coronavirus, and influenza A (19, 20) . Smokers and chronic COPD patients were reported to be more susceptible to making it a serious barrier to developing safe vaccines against these viruses. There is less high-affinity anti-COVID-19 IgG in children, which may partly explain the decreased disease severity, due to decreased ADE (4, 14, 22) .

The patients with positive chest imaging and lymphocytopenia had a higher level of SARS-CoV-2-specific IgM. Lymphocytopenia is common in COVID-19 patients and might be a critical factor associated with lung injury, disease severity, and 16 mortality (23, 24) . In COVID-19 patients, the counts of peripheral CD4 and CD8 T lymphocytes are substantially reduced, and their immuno-status is hyperactivated, indicating severe immune injury in these patients (25) .

The severe/critical cases had a higher SARS-CoV-2-specific IgM levels, and more attention should be paid to patients with IgM level >29.95 AU/ml, as they had a two-fold increased risk of deteriorating into severe/critical cases. Although natural neutralizing IgG may contribute to viral clearance to some extent (4, 26) , it is less likely that disease progression and clinical deterioration result from a depressed humoral response to SARS-CoV-2 because higher IgG levels were detected in patients with more severe disease compared to milder patients. Similar to SARS, it seems more likely that a robust humoral response to SARS-CoV-2 was one component of an overall exaggerated immune response in COVID-19, which is associated with cytokine storms (e.g., interferon [IFN]-γ and interleukin [IL]-6) (27, 28) . ADE modulates the immune response and elicits sustained systemic inflammation, lymphocytopenia, and organ dysfunction, one or all of which have been documented in many critical cases and deaths (29) . While the immunological host response to SARS-CoV-2 infection has not yet been fully elucidated to confirm whether ADE occurs, the current clinical evidence and our data suggest this is a possibility. Based on in vitro studies (30) and mouse models (31) of SARS-CoV infection, ADE decreases the ability to control inflammation in the lungs, kidneys, and elsewhere.

This mechanism may account for the acute respiratory distress syndrome and other observed inflammation-based organ injuries seen in many severe and critical COVID-19 patients (29) . Further studies need to investigate how the virus interacts with the host immune system, leading to the great variation in clinical manifestations.

There are several limitations in this study. First, this was a study on aggregate data and we lacked dynamic patient-level data, so this may have affected the results.

Second, although it was a multicenter study, the sample size was small, which weakened the strength of our findings. Third, the study duration was only 7 weeks, so the results cannot fully explain the whole immune process in humans. More large-scale, long-term clinical studies focusing on patient-level data are needed to further confirm the conclusions.

Quantitative measurements of SARS-CoV-2-specific IgM and IgG levels are helpful for the diagnosis, severity classification, and management of COVID-19

patients, and these levels should be monitored in each stage of this disease. writing of the report; or decision to submit the article for publication.

All authors declare no competing interests.

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Written informed consent was provided by the patients or their family members. This study was approved by the Ethics Committee of Zhongda Hospital, Affiliated with Southeast University (2020ZDSYLL018-P01). This article does not contain any animal studies performed by any of the authors. ECMO: extracorporeal membrane oxygenation; COPD: chronic obstructive pulmonary disease. non-mild group.

A novel coronavirus outbreak of global health concern

Emerging novel coronavirus (2019-nCoV)-current scenario, evolutionary perspective based on genome analysis and recent developments

Clinical Significance of an IgM and IgG Test for Diagnosis of Highly Suspected COVID-19

Anti-SARS-CoV IgG response in relation to disease severity of severe acute respiratory syndrome

MERS-CoV Antibody Responses 1 Year after Symptom Onset, South Korea

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Antigenic cross-reactivity between the nucleocapsid protein of severe acute respiratory syndrome (SARS) coronavirus and polyclonal antisera of antigenic group I animal coronaviruses: implication for SARS diagnosis

Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design

The spike protein of SARS-CoV--a target for vaccine and therapeutic development

Antibody responses to SARS-CoV-2 in patients with COVID-19

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites

Longitudinal profile of immunoglobulin G (IgG), IgM, and IgA antibodies against the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in patients with pneumonia due to the SARS coronavirus

SARS-CoV-2 infections in children and young people

Serial analysis of the plasma concentration of SARS coronavirus RNA in pediatric patients with severe acute respiratory syndrome

Severe acute respiratory syndrome (SARS) in neonates and children

A Well Infant With Coronavirus Disease 2019 With High Viral Load

Mutagenicity for Salmonella typhimurium of urine obtained from humans receiving nitrofurantoin

First infection by all four non-severe 21

Prevalence and Risk Factors of Respiratory Viral Infections in Exacerbations of Chronic Obstructive Pulmonary Disease

Prevalence and contribution of respiratory viruses in the community to rates of emergency department visits and hospitalizations with respiratory tract infections, chronic obstructive pulmonary disease and asthma

Middle East Respiratory Syndrome Coronavirus Receptor, is Upregulated in Lungs of Smokers and Chronic Obstructive Pulmonary Disease Patients

Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins

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

Clinical characteristics of 140 patients infected with SARS-CoV-2 in Wuhan, China

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

Neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection

An interferon-gamma-related cytokine storm in SARS patients

Characterization of cytokine/chemokine profiles of severe acute respiratory syndrome

Is COVID-19 receiving ADE from other coronaviruses

Severe acute respiratory syndrome (SARS) coronavirus-induced lung epithelial cytokines exacerbate SARS pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells

Dysregulated Type I Interferon and Inflammatory Monocyte-Macrophage Responses Cause Lethal Pneumonia in SARS-CoV-Infected Mice

2-specific IgM and IgG levels in COVID-19 patients from week 1 to 7; B. Percentage of COVID-19 patients with positive serum SARS-CoV-2-specific IgM and IgG from week 1 to 7; C. Correlation between the peak SARS-CoV-2-specific IgG level (from week 1 to 5) and the duration of positive nucleic acid test in COVID-19 patients, showing the relationship between the antibody level and virus clearing ability. A higher antibody level, regardless of the time point

Comparison of serum SARS-CoV-2-specific IgM between mild/moderate and severe/critical groups from week 1 to 7