key: cord-351662-rmkcb6o3 authors: Huang, Zhifeng; Chen, Hao; Xue, Mingshan; Huang, Huimin; Zheng, Peiyan; Luo, Wenting; Liang, Xueqing; Sun, Baoqing; Zhong, Nanshan title: Characteristics and roles of SARS‐CoV‐2 specific antibodies in patients with different severities of COVID‐19 date: 2020-07-24 journal: Clin Exp Immunol DOI: 10.1111/cei.13500 sha: doc_id: 351662 cord_uid: rmkcb6o3 BACKGROUND: The diagnosis of COVID‐19 relies mainly on viral nucleic acid detection, but false negatives can lead to missed diagnosis and misdiagnosis. SARS‐CoV‐2‐specific antibody detection is convenient, safe, and highly sensitive. IgM and IgG are commonly used to serologically diagnose COVID‐19; however, the role of IgA is not well known. We aimed to quantify the levels of SARS‐CoV‐2‐specific IgM, IgA, and IgG antibodies, identify changes in them based on COVID‐19 severity, and establish the significance of combined antibody detection. METHODS: COVID‐19 patients, divided into a severe & critical group and a moderate group, and non‐COVID‐19 patients with respiratory disease were included in this study. A chemiluminescence method was used to detect the levels of SARS‐CoV‐2‐specific IgM, IgA, and IgG in the blood samples from the three groups. Epidemiological characteristics, symptoms, blood test results, and other data were recorded for all patients. RESULTS: Compared to the traditional IgM–IgG combined antibodies, IgA–IgG combined antibodies are better for diagnosing COVID‐19. During the disease process, IgA appeared first and disappeared last. All three antibodies had significantly higher levels in COVID‐19 patients than in non‐COVID‐19 patients. IgA and IgG were also higher for severe & critical disease than for moderate disease. All antibodies were at or near low levels at the time of tracheal extubation in critical patients. CONCLUSIONS: Detection of SARS‐CoV‐2‐specific combined IgA–IgG antibodies is advantageous in diagnosing COVID‐19. IgA detection is suitable during early and late stages of the disease. IgA and IgG levels correspond to disease severity. A novel coronavirus pneumonia outbreak commenced in Wuhan, China, in late December 2019 [1] and spread rapidly throughout the country and overseas. In February 2020, the World Health Organization Accepted Article named the virus SARS-CoV-2 and the disease COVID-19. Thus far, more than 3.3 million people have been infected and 230,000 have died worldwide; these numbers are expected to rise further. The diagnosis of COVID-19 depends on the detection of the viral nucleic acid; however, the detection of SARS-CoV-2 nucleic acid has a high false negative rate, making the disease easy to misdiagnose. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect SARS-CoV-2 in 4,880 patients with COVID-19 in a hospital in Wuhan. While less than 50% of nasopharynx swabs and sputum were positive, bronchoalveolar lavage fluid had a 100% positivity rate [2] . The acquisition of alveolar lavage fluid, however, is invasive and not suitable for most patients with mild disease. Current COVID-19 diagnosis guidelines suggest the combined use of nucleic acid detection and clinical symptomology [3] . However, the large number of asymptomatic people positive for SARS-CoV-2 make this method flawed. Thus, it is necessary to increase the detection of the SARS-CoV-2-specific antibody. The detection of the SARS-CoV-2-specific antibody is convenient, safe, and highly sensitive. It has certain advantages in the auxiliary diagnosis of COVID-19. The moderately used SARS-CoV-2-specific antibodies include IgM, which signifies the primary immune response and indicates a recent infection, and IgG, which is the main antibody produced by the secondary immune response. IgA is often ignored in the diagnosis of COVID-19. Guo et al. [4] found that IgA and IgM appear simultaneously, which is important for diagnosing patients with acute or asymptomatic infection. The specific IgA antibody, therefore, should be considered in the diagnosis of COVID-19. The diagnostic efficacy of specific IgA antibody and the levels of these specific antibodies depending on disease severity are currently unclear. The rise times for specific IgM and IgG levels are different, and combined detection could be more advantageous in the diagnosis of COVID-19 [5] . Large-scale detection of SARS-CoV-2-specific IgM and IgG has been carried out nationwide, but combined evaluation is rare. The benefit of combining the detection of specific IgA with that of combined specific IgM-IgG is still uncertain. In this study, SARS-CoV-2-specific IgM, IgA, and IgG levels were measured in patients with varying severities of COVID-19, the relationship between specific antibody levels and disease severity was classified, and the significance of combined antibody detection was clarified, providing a reference for the clinical diagnosis of COVID-19. This article is protected by copyright. All rights reserved All COVID-19 patients tested positive for the SARS-CoV-2 viral nucleic acid. The clinical classification of COVID-19 was determined according to the Guidelines of the Diagnosis and Treatment of New Coronavirus Pneumonia (version 7) published by the National Health Commission of China [6] . Moderate disease was characterized by fever, respiratory and other symptoms, and the manifestation of viral pneumonia on computed tomography (CT) imaging. Severe disease met at least one of the additional following conditions: (1) shortness of breath with respiratory rate ≥30 times/min, (2) 44, 2020). Written informed consent was waived in light of this emerging infectious disease of high clinical relevance. All healthy control subjects signed written informed consent prior to the collection of peripheral blood. Forty-nine days after symptom onset, 298 serum samples from 43 COVID-19 patients were collected. At the first visit in Respiratory Clinic of the 61 non-COVID-19 patients, 61 serum samples were collected. All serum samples were inactivated in a water bath at 56 ºC for 30 minutes. KAESER 6600 automatic chemiluminescence immunoanalyzer and matching reagents kit (Guangzhou Kangrun Biotech Co., Ltd.) was used to detect the SARS-CoV-2-specific IgM, IgA, and IgG levels using a two-step indirect detection method. A sequence encoding receptor binding region of spike protein (S protein) was cloned into pTT5 vector, and the constructed expression vector was used to transiently transfect HEK293F cells. The carboxyl group on the magnetic beads is activated by an activator, and then the amino group on the S protein is coupled with the carboxyl group of the magnetic beads to form an amide bond, and the antigen is fixed on the magnetic beads. Anti-human IgA, M, G antibodies were coupled with acridine ester derivatives. The specific antibody in the testing sample was combined with a magnetic bead coating (S protein recombinant antigen) to form a magnetic bead coating-specific antibody complex. After the unbound substances were separated and washed with magnetic beads, the acarithrate marker was added to form the magnetic bead coating material-SARS-CoV-2-specific antibody-acarithrate labeling complex. A photomultiplier was used to detect light signals from acridine ester that were converted to obtain the corresponding signal value. The relative light signal values, expressed in relative light units (RLU), indicated IgM, IgA, and IgG levels. The relative light signal value is equivalent to the original signal value over the specific antibody cut-off value. The cut-off values of IgM, IgA, and IgG are 11,300, 56,492, and 42,213, respectively. A relative luminescence value (RLV) greater than or equal to 1.0 is positive for specific IgM, IgA, and IgG. Moderately distributed continuous data are represented as means and standard deviations, while non-moderately distributed data are indicated by medians and interquartile ranges (IQR). The chi-squared test or Fisher's exact probability test was used to compare qualitative data. The Mann-Whitney U test was used for independent sample comparison between two groups of non-parametric data. The Kruskal-Wallis H test was used for comparison between multiple groups. For statistical purposes, we grouped severe & critical patients. P <0.05 was considered statistically significant. SPSS 23.0 (IBM Corp., Armonk, NY, USA) and Graphpad Prism 8.0.1 (© 1995-2020 GraphPad Software, LLC) were used for data analyses. This article is protected by copyright. All rights reserved Among the severe & critical COVID-19 patients, nine were severe and ten were critical. Of all included patients, 28 were male and 15 were female. The average ages of the patients with severe & critical disease and moderate disease were 50 and 60 years, respectively. There was no statistically significant difference between the ages of the two groups. The severe & critical group had significantly more exposure history in Wuhan than the moderate group (P<0.05). COVID-19 patients had significantly more symptoms of fever, wheezing, and fatigue than non-COVID-19 patients (P<0.01). The leukocyte count of the severe & critical and non-COVID-19 groups were significantly higher than that of the moderate group (P<0.01). The lymphocyte and basophil count of the severe & critical group were significantly lower than those of the non-COVID-19 group (P<0.01). The chest CTs of COVID-19 patients all revealed suspected viral pneumonia. Interestingly, ten non-COVID-19 patients reported the same CT findings. Throughout the entire disease course, specific IgM, IgA, and IgG were detected in almost all patients with COVID-19. In addition, ten non-COVID-19 patients had positive IgM and two had positive IgA. This article is protected by copyright. All rights reserved This article is protected by copyright. All rights reserved We compared the distribution of IgA, IgG, and IgM antibodies between COVID-19 and non-COVID-19 patients (Figure 3) . The levels of the three antibodies were significantly higher in COVID-19 patients than in non-COVID-19 patients (P<0.01). In addition, the level of IgA ( in moderate COVID-19 patients. All differences were statistically significant (P<0.01). There was no statistically significant difference in the level of IgM, however, between the severe & critical and moderate groups. Ten critical COVID-19 patients were included in our study. By data collection, six of the patients had been extubated. Tracheal extubation was used as a marker of symptom improvement in critical COVID-19 patients. We found that the six extubated patients had undergone endotracheal tube removal on an average of 30 days after symptom onset. IgA, IgG, and IgM were at or near low levels at the time of tracheal extubation (Figure 4 ). The COVID-19 outbreak has caused a great loss of life across the world. The gold standard of diagnosis, detection of SARS-CoV-2 nucleic acid by RT-PCR, is prone to missed diagnoses due to its false negative rate. To some extent, the detection of the SARS-CoV-2-specific antibody can make up for a deficiency in nucleic acid detection. Studies [7] have shown that in suspected COVID-19 cases with negative nucleic acid, the specific antibody is an effective supplementary indicator of SARS-CoV-2 infection and can be combined with nucleic acid testing to confirm infection. Xie et al. also believe that the combination of nucleic acid and the IgM-IgG antibody test is the optimal method for diagnosing SARS-CoV-2 infection [8] .This provides a more convenient and fast method for the diagnosis and avoidance of missed diagnoses of COVID-19. In our study, the included patients were divided into three groups: severe & critical COVID-19, moderate This article is protected by copyright. All rights reserved COVID-19, and non-COVID-19 with respiratory diseases. Compared with moderate COVID-19 patients, severe & critical COVID-19 patients were more likely to have a history of Wuhan exposure, consistent with the clinical characteristics of COVID-19 reported in the Hunan and Anhui provinces [9, 10] . COVID-19 patients were more likely than non-COVID-19 patients to develop fever, wheezing, and fatigue, the same COVID-19 symptoms initially reported in Wuhan [11] and now established as the typical symptoms. In IgM and IgG have a reciprocal relationship; therefore, the simultaneous detection of IgM and IgG antibodies is more suitable for COVID-19 patients with an unclear infection stage. In a study evaluating the sensitivity and specificity of IgG and IgM combined antibodies [18], the combined antibodies showed 88.66% sensitivity and 90.63% specificity in the diagnosis of COVID-19. This was superior to IgM or IgG testing alone. We found that IgA-IgG combined detection, however, is better than the traditional IgM-IgG combined detection, preventing missed diagnosis and misdiagnosis to a greater extent. In all included COVID-19 patients, IgA increased in the first week after symptom onset, peaked in the third week, and gradually decreased thereafter. IgM levels in severe & critical COVID-19 patients remained high in the second week, and in moderate COVID-19 patients, it remained high in the first week after symptom onset and peaked in the fourth week before slowly decreasing. IgG levels in severe & critical COVID-19 patients remained high in the second week, peaked in the fourth week, then began to decline, and increased again in the sixth week. In moderate COVID-19 patients, IgG levels increased in the first week and peaked in the third week. Guo et al. [4] found that after SARS-CoV-2 infection in humans, specific antibodies are produced in 1-5 days, specific IgM and IgA are detected in 3-6 days, and IgM levels rise to the highest level in 8-14 days after symptoms appear. The IgA level continues to rise for 0-14 days following symptom onset and thereafter ceases to increase. The IgG can be detected 14 days after the onset of symptoms, rises during days 8-21, stabilizes after 21 days, and remains present in the later stages of infection. Andrea P et al. [19] found that the IgA response appears early, peaks at week 3, and it is stronger and more persistent than the IgM response, which was similar to our results. In our study, however, the peak of IgM, IgA, and IgG levels were detected one week after they were detected in the study by Guo et al. [4] . We also found that while IgA and IgG levels were significantly higher in the severe & critical patients than in moderate patients, there was no difference in IgM between the two groups. These results suggest that while IgA and IgG could reflect disease severity, IgM did not have the same quality. In order to further observe the relationship between SARS-CoV-2-specific antibodies and COVID-19 severity, we considered tracheal extubation to be an index of improvement in critical COVID-19 patients. We found that the specific antibodies were at or near low levels at the time of tracheal extubation, indicating that dynamic This article is protected by copyright. All rights reserved monitoring of SARS-CoV-specific antibodies may help determine the optimal time for extubation and help guide the treatment of critical patients. More cases must be studied and other factors that may contribute to bias should be ruled out before accurate conclusions can be drawn. There are some limitations to our study. First, only a small number of cases were included with limited clinical data in moderate COVID-19 and non-COVID-19 patients. Second, almost all severe & critical COVID-19 patients were transferred from other hospitals; therefore, antibody levels during the first week after symptom onset could not be monitored. Since we also were unable to monitor the antibody levels of patients after discharge, we did not fully understand the dynamic trend of antibody levels four and seven weeks after symptom onset in moderate and severe & critical COVID-19 patients, respectively. In conclusion, compared to the traditional detection of IgM-IgG combined antibodies, the detection of SARS-CoV-2-specific IgA-IgG combined antibodies is more advantageous in the diagnosis of COVID-19. SARS-CoV-2-specific IgA detection is even more suitable than IgM detection in the early stages of COVID-19 and has important reference value in the later stages of the disease. Levels of IgA and IgG were higher in severe & critical COVID-19 patients than in moderate COVID-19 patients, while IgM levels were no different between the two groups. This suggests that IgA and IgG levels are associated COVID-19 severity; therefore, in the serological diagnosis of COVID-19 using SARS-CoV-2-specific IgM and IgG, we suggest that more attention should be paid to specific IgA levels. Funding: Zhejiang University special scientific research funding for COVID-19 prevention and control (2020XGZX001, 2020XGZX025). This article is protected by copyright. All rights reserved Note: # The numbers of both or any one antibodies positive / total numbers. * The numbers of both of the two antibodies negative / total numbers. Po, positive; Ne, negative. A Novel Coronavirus from Patients with Pneumonia in China Positive rate of RT-PCR detection of SARS-CoV-2 infection in 4880 cases from one hospital in Combination of RT-qPCR testing and clinical features for diagnosis of COVID-19 facilitates management of SARS-CoV-2 outbreak Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19) Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 Guidelines for the Diagnosis and Treatment of Novel Coronavirus (2019-nCoV) Infection by the National Health Commission Evaluation of the auxiliary diagnostic value of antibody assays for the detection of novel coronavirus (SARS-CoV-2) Characteristics of patients with coronavirus disease (COVID-19) confirmed using an IgM-IgG antibody test Clinical characteristics of 161 cases of corona virus disease 2019 (COVID-19) in Changsha Initial clinical features of suspected coronavirus disease 2019 in two emergency departments outside of Hubei Clinical features of patients infected with 2019 novel coronavirus in Wuhan Accepted Article We have to thank all the patients we included, and to thank qingyuan and yangjiang people's hospital for providing us with blood samples and data of COVID-19 patients. The data that support the findings of this study are available from the *** *** *** cei_13500_f3.eps This article is protected by copyright. All rights reserved Accepted Article This article is protected by copyright. All rights reserved corresponding author upon reasonable request.