key: cord-1001261-c2v4k164
authors: Xie, Jiajia; Ding, Chengchao; Li, Jing; Wang, Yulan; Guo, Hui; Lu, Zhaohui; Wang, Jinquan; Zheng, Changcheng; Jin, Tengchuan; Gao, Yong; He, Hongliang
title: Characteristics of patients with coronavirus disease (COVID‐19) confirmed using an IgM‐IgG antibody test
date: 2020-05-07
journal: J Med Virol
DOI: 10.1002/jmv.25930
sha: 1b5ce86bb8c30a7bd184183e26d30d6da8ca56d9
doc_id: 1001261
cord_uid: c2v4k164

Coronavirus disease (COVID‐19), caused by a novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), has rapidly developed into a pandemic since it was first reported in December 2019. Nucleic acid testing is the standard method for the diagnosis of viral infections. However, this method reportedly has a low positivity rate. To increase the sensitivity of COVID‐19 diagnoses, we developed an IgM‐IgG combined assay and tested it in patients with suspected SARS‐CoV‐2 infection. In total, 56 patients were enrolled in this study and SARS‐CoV‐2 was detected by using both IgM‐IgG antibody and nucleic acid tests. Clinical and laboratory data were collected and analyzed. Our findings suggest that patients who develop severe illness might experience longer virus exposure times and develop a more severe inflammatory response. The IgM‐IgG test is an accurate and sensitive diagnostic method. A combination of nucleic acid and IgM‐IgG testing is a more sensitive and accurate approach for diagnosis and early treatment of COVID‐19.

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first emerged in Wuhan, China, on 12 December 2019, it has spread quickly across the world and developed into a pandemic. [1] [2] [3] The World

Health Organization (WHO) announced a new name for the disease:

coronavirus disease (COVID-19). By 31 March 2020, more than 700 000 COVID-19 cases were confirmed in over 100 countries and regions. To date, the rapid spread of SARS-CoV-2 has caused considerable harm to public health and the economy. 4, 5 Clinical manifestations of COVID-19 include fever, dry cough, and fatigue. Approximately half of the infected patients developed severe pneumonia, and nearly one-third of the patients develop acute respiratory distress syndrome. [6] [7] [8] However, there is currently no specific treatment for COVID-19. Due to the obstacle of collecting high-quality throat swab samples in different stages of infection, nucleic acid test for SARS-CoV-2 showed high false negative rate. It is difficult to identify and quarantine the infected individuals to effectively break the spread chain and curb the infection. Therefore, it is of urgent need to develop a more sensitive diagnostic method that can rapidly identify SARS-CoV-2 infected patients with high accuracy. Jiajia Xie and Chengchao Ding contributed equally to this work.

Currently, the viral nucleic acid real-time polymerase chain reaction (RT-PCR) test based on patient nasopharyngeal and throat swabs is the standard method for clinical diagnosis of COVID-19.

Despite its crucial role in identifying SARS-CoV-2 infection in patients at the start of the epidemic, the limitations of this method soon became obvious. For example, one recent study demonstrated that RT-PCR only showed a positive test rate of 38% in a total of 4880 specimens with a significant number of false negative cases. 9 It is accepted that IgM is the early immunoglobulin in response to the virus invasion and IgG has the highest opsonization and neutralization activities in humoral immune response. Previous studies have reported that IgM-IgG seroconversion can start as early as 4 days after the onset of SARS infection. 10 Most patients were admitted to the hospital due to fever or respiratory symptoms. Nasopharyngeal and throat swabs were used for respiratory pathogen testing. Serum levels of IgM-IgG antibodies targeting SARS-CoV-2 were tested upon patient admission. Medical history about when the clinical symptoms appeared was asked and the time interval between clinical symptoms and antibody testing was recorded in detail. Physical findings and hematological and biochemical results were also collected. All patients enrolled in this study were diagnosed according to the 5th edition of the Guideline on diagnosis and treatment of COVID-19 established by China's National Health Commission, including patient's epidemic history, clinical characteristics, chest computed tomographic (CT) scan, and laboratory findings. Patients with COVID-19 having severe illness were defined having one of the following criteria: (a) respiratory distress with respiratory frequency (RP) more than or equal to 30/min, (b) pulse oximeter oxygen saturation less than or equal to 93% at rest, or (c) oxygenation index (arterial partial pressure of oxygen/inspired oxygen fraction [PaO 2 /FiO 2 ]) less than or equal to 300 mm Hg.

Clinical characteristics were compared between severe and nonsevere cases. This study was approved by the Ethics Committee of the First Affiliated Hospital of USTC. This is a retrospective and observational study and the informed consent was obtained.

The presence of SARS-CoV-2 was detected using RT-PCR. Viral RNA was extracted from nasopharyngeal and throat swabs using the 

Anti-human IgG and IgM assays were purchased from YHLO Biological Technology Co, Ltd, Shenzhen, China. In all patients, IgG and IgM antibodies against the SARS-CoV-2 envelope (E) protein and nucleocapsid (N) protein in serum samples were measured using chemiluminescence immunoassay. The cutoff value for a positive result was 10, and samples with values greater than or equal to 10 AU/mL were considered positive for SARS-CoV-2 infection.

Categorical variables are presented as numbers (%) and continuous measurements as medians (interquartile range [IQR]). Antibody concentration was reported as the geometric mean (SD). Continuous variables were analyzed using the Mann-Whitney test or unpaired t test. The correlation of IgM and IgG quantitative detection with hematological profiles was analyzed using Pearson correlation. Graphpad Prism 8.3 was used for all statistical analyses. A two-sided α value less than .05 was considered statistically significant.

IgM-IgG antibody levels and nucleic acid test results are summarized in (Table 2) .

Interestingly, there were differences in laboratory findings between the groups with severe and nonsevere COVID-19 symptoms.

This included higher neutrophil counts, neutrophil percentage (NEU%), and fibrinogen level, and lower lymphocyte counts and lymphocyte percentages (LYM%) (P < .05) (Figure 1 ). The median D-dimer level was increased in the group with severe symptoms, but the difference was not significant. Procalcitonin and hypersensitive C-reactive protein levels were in the normal range in the majority of patients (Table 3) . ). Statistical analysis was performed using the Mann-Whitney test. P values indicate differences between severe and nonsevere patients. *P < .05 was considered statistically significant. Abbreviation: COVID-19, coronavirus disease. *P < .05. **P < .005. ***P < .001. ****P < .0001. (Table 4 ; Figure 2D ).

In this study, we analyzed the clinical features and immunological characteristics of 56 patients with COVID-19. Despite negative nucleic acid test results, all patients showed high specific IgG concentrations, suggesting SARS-CoV-2 infection. Of the 56 patients, over 50% developed severe illness and required intensive care.

Common symptoms were fever, cough, and chest tightness, which is consistent with previous studies. 4, 6, 12 Compared with patients with nonsevere symptoms, patients in the severe illness group had numerous laboratory abnormalities, such as higher neutrophil counts, NEU%, fibrinogen levels, lower lymphocyte counts, and lower LYM%.

IgM was lower while IgG was higher in patients with severe symptoms. In addition, a weak correlation between IgM and NEU% was noted. These findings suggest that patients in the severe illness group might experience a longer virus exposure time and develop a more severe inflammatory response.

Currently, the nucleic acid test based on individual nasopharyngeal and throat swabs is the standard diagnostic method for COVID-19. Although the RT-PCR method is sensitive and effective, it still suffers from some limitations such as being labor-intensive F I G U R E 2 Analysis of IgM-IgG findings in severe and nonsevere groups. A, Kinetic analysis of IgM-IgG antibodies in severe and nonsevere groups; B, differences in total IgM levels between severe and nonsevere groups; C, differences in total IgG levels between severe and nonsevere groups; and D, correlation between IgM and NEU% in patients in the severe group. Antibody concentration was presented as the geometric mean (SD) and analyzed using an unpaired t test. The correlation of IgM-IgG with hematological profiles was analyzed using Pearson correlation. All statistical analyses were performed using GraphPad Prism 8.3 (****P < .0001). P < .05 was considered statistically significant. NEU%, neutrophil percentage; SD, standard deviation 

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All the authors declare that there are no conflict of interests.

HH and YG secured the funding for this study, designed the study, participated in data analysis, and extensively reviewed the manuscript. JX and CD analyzed the data and drafted the manuscript.Other authors contributed to clinical and laboratory data acquisition and reviewed the manuscript.

http://orcid.org/0000-0002-9759-9931Chengchao Ding http://orcid.org/0000-0002-4058-2725