key: cord-318615-uhh3owcx authors: Xiang, Fei; Wang, Xiaorong; He, Xinliang; Peng, Zhenghong; Yang, Bohan; Zhang, Jianchu; Zhou, Qiong; Ye, Hong; Ma, Yanling; Li, Hui; Wei, Xiaoshan; Cai, Pengcheng; Ma, Wan-Li title: Antibody Detection and Dynamic Characteristics in Patients with COVID-19 date: 2020-04-19 journal: Clin Infect Dis DOI: 10.1093/cid/ciaa461 sha: doc_id: 318615 cord_uid: uhh3owcx BACKGROUND: The corona virus disease 2019 (COVID-19) caused by the corona virus 2 (SARS-CoV-2) has been rapidly spreading nationwide and abroad. A serologic test to identify antibody dynamics and response to SARS-CoV-2 was developed. METHODS: The antibodies against SARS-CoV-2 were detected by an enzyme-linked immunosorbent assay (ELISA) based on the recombinant nucleocapsid protein of SARS-CoV-2 in patients with confirmed or suspected COVID-19 at 3-40 days after symptom onset. The gold standard for COVID-19 diagnosis was nucleic acid testing for SARS-CoV-2 by RT-PCR. The serodiagnostic power of the specific IgM and IgG antibodies against SARS-CoV-2 was investigated in terms of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and consistency rate. RESULTS: The seroconversion of specific IgM and IgG antibodies were observed as early as the 4(th) day after symptom onset. In the confirmed patients with COVID-19, sensitivity, specificity, PPV, NPV, and consistency rate of IgM were 77.3% (51/66), 100%, 100%, 80.0%, and 88.1%, and those of IgG were 83.3.3% (55/66), 95.0%, 94.8%, 83.8%, and 88.9 %. In patients with suspected COVID-19, sensitivity, specificity, PPV, NPV, and consistency rate of IgM were 87.5% (21/24), 100%, 100%, 95.2%, and 96.4%, and those of IgG were 70.8% (17/24), 96.6%, 85.0%, 89.1%, and 88.1%. Both antibodies performed well in serodiagnosis for COVID-19 rely on great specificity. CONCLUSIONS: The antibodies against SARS-CoV-2 can be detected in the middle and later stage of the illness. Antibody detection may play an important role in the diagnosis of COVID-19 as complement approach for viral nucleid acid assays. A novel corona virus (SARS-CoV-2) disease (COVID-19) was first identified and outbroke in Wuhan City, Hubei Province, China. A total of 93, 090 patients with COVID-19 had occurred globally by March 5, 2020 [1] . Accurate and fast diagnosis of the causative SARS-CoV-2 is important to isolate the patients with COVID-19 timely and stop the epidemics, as well as save people's lives. Viral nucleic acid detection using real-time polymerase chain reaction (RT-PCR) assay, which has been developed and used for detection of SARS-CoV-2 rapidly, remains the standard diagnosis of COVID-19 [2] . While a large number of the "suspected" cases with typical clinical COVID-19 features and/or identical specific computed tomography (CT) scan were not diagnosed [3, 4] . Moreover, RT-PCR assay which was time consuming and laborious needed special equipment resulted to limit its usage especially in remote areas. The human antibody response which is crucial for the clearance of the initial virus infection has been widely used to help diagnosis virus infection. Compared to RT-PCR assays, the detection of antibody assays are often faster, less expensive, easy-to-use and accessible to staff without laboratory training. Here, we detected dynamics characteristics and magnitude of antibody response in patients with COVID-19, and evaluated serodiagnostic value of ELISA-based IgM, IgG tests for COVID-19 pneumonia. The sensitivity and specificity of antibody tests for detection of IgM and IgG were presented and the clinical application of these antibody assays for serodiagnosis of COVID-19 was discussed. Patients at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology were evaluated from January 19, 2020 to March 2, 2020. During this period, IgM and IgG antibody responses to SARS-CoV-2 virus infection were analyzed. The gold standard for diagnosis is nucleic acid testing for SARS-CoV-2 by RT-PCR tests of nasopharyngeal and/or oropharyngeal swabs sample. RT-PCR tests were performed for patients who presented with a history of 1) Travel or residential history in Wuhan or local endemic areas; or 2) The epidemioloical history of contact with patients who have been confirmed with COVID-19 pneumonia or individuals present fever or respiratory symptoms from these areas within 14 days; or 3) A clustering outbreak, combined with clinical manifestation of 1) fever and /or respiratory symptoms, or 2) positive findings similar to COVID-19 pneumonia on chest computerized tomography (CT) scan, or 3) laboratory tests showing reduced lymphocytes and white blood cell counts in the early stage. In the case of an initial negative RT-PCR test, repeated testing was performed at intervals of one day or more. The diagnosis of laboratory confirmed COVID-19 defined as positive nucleic acid tests for SARS-CoV-2 by RT-PCR assays. The diagnosis of suspected COVID-19 was based on one of the epidemiological history and two of the clinical manifestations but the RT-PCR of SARS-CoV-2 was negative. In confirmed cases, the patient showing fever and respiratory symptoms with radiological findings of pneumonia was defined as normal case, while the case meeting any of the following criteria was defined as severe case: 1) Respiratory distress (≧30 breaths/ min); 2) Oxygen saturation≤93% at rest; 3) Arterial partial pressure of oxygen (PaO2)/fraction of inspired oxygen (FiO2) ≤ 300mmHg (l mmHg=0.133kPa); 4) Cases with chest imaging that shows obvious lesion progression within 24-48 hours >50% shall be 6 managed as severe cases. In the control group, samples from healthy blood donors or from patients with other disease hospitalized in the same hospital. The detailed diagnosis standards were presented in online Table S1 . Total RNAs were extracted from nasopharyngeal and/or oropharyngeal swabs samples of patients suspected of having SARS-CoV-2 infection within 2 hours using the respiratory sample RNA isolation kit. In brief, 40 μL of cell lysates were transferred into a collection tube followed by vortex for 10 seconds. After standing at room temperature for 10 minutes, the collection tube was centrifuged at 1000 rpm/min for 5 minutes. The suspension was used The serum SARS-CoV-2 antibodies (IgM and IgG) of the subjects were detected using a sandwich enzyme linked immunosorbent assay (ELISA kits, Livzon Inc, Zhuhai, P.R.China, lot number of IgM: 20200308, IgG: 20200308). For detection of IgM, 100 µl diluted serum (1:100) was added into the 96-well microplate (coated with N protein) and then incubated for 1 h at 37℃. After washing, 100 µl secondary antibody (against human IgM) labeled with conjugate was added into the wells and then incubated for 30 min under 37℃. Following the second wash cycle, 100 µl substrate was added into the wells and incubated for 15 min under 37℃. At last, stop solution was added into the wells to terminate the reaction. The optical density of each well was determined by a microplate reader set to 450 nm within 30 min. The ratio of optical density to the cut off value (optical density of the blank well + 0.1) was reported as the antibody concentration. For detection of IgG, the dilution factor was changed (1:20) and the cut off value was modified (optical density of the blank well + 0.13). All statistical analyses were performed using SPSS 20.0. The diagnostic value of ELISAbased IgM and IgG antibody test for COVID-19 was based on sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy. Log-normal distribution was used to fit the time distribution of IgM and IgG antibody seropositve rate, as well as calculative seroconversion rate of the two antiobdies. 8 Ethics approval was exempted from institutional review board of the hospital since we collected and analyzed all data from the patients according to the policy for public health outbreak investigation of emerging infectious diseases issued by the National Health Commission of the People's Republic of China. 85 patients with confirmed diagnosis and 24 patients with suspected diagnosis were recruited in this study. The demographic and clinical characteristics were showed in Tabel 1. Comorbidities in patients were recorded including hypertension, diabetes, surgical operation, malignancies, chronic lung disease, and chronic renal diseases. Healthy donors who were doctors and nurses working in the hospital, and patients with other lung disorders including bacterial pneumonia (n=5), acute exacerbation of chronic obstructive pulmonary disease (AECOPD, n=2), lung cancer (n=3), empyema (n=1), interstitial lung disease (n=2) and pleuritis (n=1) were included as control group. Detection of IgM and IgG Antibody on Different Periods (Table 2.) We evaluated specificity of IgM and IgG antibodies based on ELISA from 216 serum sample of 85 confirmed COVID-19 pneumonia patients. Serum obtained in different periods after 9 symptom onset. The IgM and IgG antibodies were detected positive as early as on the 4 th day after onset (Table 2) . Some patients were observed positive for IgM (7 patients) and IgG (6 patients) within 7 days after illness onset, respectively. The seropositive rate of IgM increased gradually and notably. IgG was increased sharply on the 12 th day after onset. We used a log-normal distribution to fit the time distribution of IgM and IgG antibody seropositive rate (Figure 1 ). The results from the 60 samples in the control group showed that 3 cases were positive for IgG, while all cases were negative for IgM. To monitor the kinetics of serological antibodies within COVID-19 patients, serological antibodies were tested consecutively once the initiative appearance time of IgM and/or IgG antibody were detected in 29 confirmed patients. A log-distribution was used to fit the seroconversion time of the two antibodies ( Figure 2 ). IgM cumulative seroconversion increased quickly from the 9 th day as well as IgG increased from the 11 th day after symptom onset. In our investigation, both antibodies were seropositive in nearly all the patients within the illness course for more than 30 days. To evaluate the diagnostic potential of serological IgM and IgG antibodies detection, 66 patients with confirmed COVID-19 pneumonia were evaluated and compare to standard RT-PCR assays. Serum sample were obtained from patients with disease course being or more than 13 days and less than 29days. The results of serological test for IgM and IgG were showed in Table 3 . Compared with RT-PCR, the sensitivity, specificity, PPV, NPV, and As shown in Table 4 , 24 patients had COVID-19 pneumonia manifestations while being negative at least twice for respiratory tract nucleic acid tests. Patients were evaluated independently by two experienced physicians. The serological tests for IgM and IgG showed in Table 5 During the immune responses against pathogens infection, IgM are usually produced earlier than IgG antibody. However, both IgM and IgG antibodies against SARS-CoV-2 were detected as early as the 4 th day after symptom onset, and the appearance of IgM and IgG antibodies seems earlier than SARS, another severe coronavirus pneumonia [9] . In our study, the seropositive rate of IgG was observed decreased around the 28 th days after illness onset. The seropositive rate should not decrease on this time point, we thought this may be due to the small sample size, there were only 7 serum samples collected from 7 patients. Diagnostic value of serological test was evaluated in patients with confirmed and suspected COVID-19 pneumonia. Depending on patients whose disease course (>=13 days from the disease onset), specificity and positive predictive value of IgM antibody were very high up to 100%, which indicated that the IgM can be used as a good mark for diagnosis of COVID-19. However, the sensitivity, negative predictive value and consistency rate of IgM were 73.2%, 80.0% and 88.1%, respectively, indicating that acute infection may still be missed based on seronegative IgM. The specificity negative predictive value and consistency rate of IgG were 95.0%, 94.8% and 88.9%, respectively. That means patients can be diagnosed as COVID-19 pneumonia base on seropositive IgG. Both seropositive antibodies demonstrate outstanding specificity and PPV, it suggest that seropositve IgM and/or IgG can help to establish the diagnosis of COVID-19 pneumonia, especially in patients with a long course. To avoid misdiagnosis, patients with early seronegative antibodies should be retested after 10 days of onset. The data acquired from patients with suspected diagnosis demonstrate that the serological tests are reliable as they show high specifity, of which IgM tests were up to 100% and IgG tests were 95.0%. Those testing results were in accordance with physician's judgments of the symptoms. There were 3 controls out of 60 were positive in IgG tests. These 3 controls (one was weak positive) were healthcare providers, they didn't serve patients who diagnosed as confirmed or suspected COVID-19 at that time. We think these should be false positive or community asymptomatic infection. In summary, detection of antibodies against COVID-19 based ELISA appears to be a valid approach to serodiagnosis of COVID-19 pneumonia. The specific circulating antibody can be uniformly detected, therefore avoiding false-negative results due to sampling or potential absence of viruses in the respiratory system. As an emerging infection disease, the current population is generally susceptible to it and the background levels of serum specific antibodies are low. Therefore, COVID-19 pneumonia can be diagnosed base on seropositive of specific antibodies as an alterative to viral nucleic acid detection with clear advantages. The authors thank Dr. Xiang-Ping Yang at Department of School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology for his review of our manuscript This work is funded by National Natural Science Foundation of China (No. 81973990, 91643101), and Science Foundation of Conflict of Interest. The authors declare that there are no conflicts of interest Coronavirus disease 2019 (COVID-19) Situation Report-44. MAR 04 2020. WHO Real-Time RT-PCR Panel for Detection 2019-Novel Coronavirus Sensitivity of Chest CT for COVID-19: Comparison to RT-PCR. Radiology Correlation of Chest CT and RT-PCR Testing in Coronavirus Disease 2019 (COVID-19) in China: A Report of 1014 Cases A novel coronavirus from patients with pneumonia in China Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China SARS and common viral infections Detection of Specific Antibodies to Severe Acute RespiratorySyndrome (SARS) Coronavirus Nucleocapsid Protein forSerodiagnosis of SARS Coronavirus Pneumonia A novel coronavirus associat ed with severe acut e respirat ory syndrome * Specific IgM and IgG antibodies against SARS-CoV-2 from 216 serum samples of 85 confirmed COVID-19 pneumonia cases were tested with ELISA. Serum samples were obtained at different time points after symptom onset