key: cord-0693378-dt7ql20m authors: Zhao, Juanjuan; Yuan, Quan; Wang, Haiyan; Liu, Wei; Liao, Xuejiao; Su, Yingying; Wang, Xin; Yuan, Jing; Li, Tingdong; Li, Jinxiu; Qian, Shen; Hong, Congming; Wang, Fuxiang; Liu, Yingxia; Wang, Zhaoqin; He, Qing; Li, Zhiyong; He, Bin; Zhang, Tianying; Fu, Yang; Ge, Shengxiang; Liu, Lei; Zhang, Jun; Xia, Ningshao; Zhang, Zheng title: Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 date: 2020-03-28 journal: Clin Infect Dis DOI: 10.1093/cid/ciaa344 sha: 80dd29275b108a285e87d758696af727a28a8318 doc_id: 693378 cord_uid: dt7ql20m BACKGROUND: The novel coronavirus SARS-CoV-2 is a newly emerging virus. The antibody response in infected patient remains largely unknown, and the clinical values of antibody testing have not been fully demonstrated. METHODS: A total of 173 patients with SARS-CoV-2 infection were enrolled. Their serial plasma samples (n=535) collected during the hospitalization were tested for total antibodies (Ab), IgM and IgG against SARS-CoV-2. The dynamics of antibodies with the disease progress was analyzed. RESULTS: Among 173 patients, the seroconversion rate for Ab, IgM and IgG was 93.1%, 82.7% and 64.7%, respectively. The reason for the negative antibody findings in 12 patients might due to the lack of blood samples at the later stage of illness. The median seroconversion time for Ab, IgM and then IgG were day-11, day-12 and day-14, separately. The presence of antibodies was <40% among patients within 1-week since onset, and rapidly increased to 100.0% (Ab), 94.3% (IgM) and 79.8% (IgG) since day-15 after onset. In contrast, RNA detectability decreased from 66.7% (58/87) in samples collected before day-7 to 45.5% (25/55) during day 15-39. Combining RNA and antibody detections significantly improved the sensitivity of pathogenic diagnosis for COVID-19 (p<0.001), even in early phase of 1-week since onset (p=0.007). Moreover, a higher titer of Ab was independently associated with a worse clinical classification (p=0.006). CONCLUSIONS: The antibody detection offers vital clinical information during the course of SARS-CoV-2 infection. The findings provide strong empirical support for the routine application of serological testing in the diagnosis and management of COVID-19 patients. Since early December of 2019 and up to March 22, 2020, over 260,000 cases of coronavirus disease 2019 (COVID-19) caused by novel coronavirus (SARS-CoV-2) infection, with over 11,000 deaths have been reported in 184 countries. 1 The World Health Organization declared that COVID-19 can be characterized as a pandemic on March 11, 2020. According to recent reports, most of COVID-19 patients have an incubation period of 3 to 7 days. 2 Fever, cough and fatigue are the most common symptoms, whereas nasal congestion, runny and diarrhea are only noted in a small part of the patients. 3 Severe cases might rapidly progress to acute respiratory distress syndrome (ARDS), septic shock and difficult-to-tackle metabolic acidosis and bleeding and coagulation dysfunction. 4 It should be noted that some of COVID-19 patients only had mild atypical symptoms initially, even for severe and critical cases. 5 The chest computed tomography of COVID-19 patients were characterized by the ground-glass opacity and bilateral patchy shadowing. 6 For laboratory test, it was reported that most of patients had lymphopenia and elevated C-reactive protein. 7 However, the above-mentioned clinical and laboratory characteristics are not easily distinguishable from pneumonia induced by infection with other common respiratory tract pathogens. The timely and accurate diagnosis of the SARS-CoV-2 infection is the cornerstone of the efforts to provide appropriated treatment for patients, to limit further spread of the virus and ultimately to eliminate the virus from human society. Currently, PCR-based viral RNA detection is almost the only way to confirm the diagnosis of SARS-CoV-2 infection in practice. On the other hand, RNA testing based on throat or nasopharyngeal swabs brought out negligible false-negative risk. 8 The reported positive rate varied for different swab specimens in COVID-19 patients. 3, 9 Many cases that were strongly epidemiologically linked to SARS-CoV-2 exposure and with typical lung radiological findings remained RNA negative in their upper respiratory tract samples. The performance of RT-PCR depends on many factors such as the sample types 9 , different stage of infection in patients, 10 the skill of sample collection, and the quality and consistency of the PCR assays being used. These problems lead to a noteworthy delay of early diagnosis and following management and propose serious challenge to providing timely life support treatment and preventive quarantine. Comparing to PCR, serological testing is advantageous with faster turn-around time, highthroughput and less workload. However, the clinical value of antibodies largely depends on the understanding of host antibody responses during infection. Given that SARS-CoV-2 is a newly emerging virus, the antibody response in COVID-19 patients remains largely unknown. This study investigates the dynamics of total antibody (Ab), IgM and IgG antibody against SARS-CoV-2 in serial blood samples collected from 173 confirmed COVID-19 patients and provides discussion on the clinical value of antibody testing. A confirmed COVID-19 case and the clinical classification was defined based on the New Coronavirus Pneumonia Prevention and Control Program (4th edition) published by the National Health Commission of China. Briefly, patient with acute respiratory infection syndromes and/or abnormalities in chest CT images accompanied by detectable SARS-CoV-2 RNA in respiratory sample since illness onset for at least one time was confirmed as COVID-19 case. Patients were classified as in critical illness condition with ARDS or oxygen saturation <93% who required mechanical ventilation either invasively or non-invasively. This study enrolls a total of 173 cases of COVID-19, where all patients were admitted to the Shenzhen Third People's Hospital between Jan 11 and Feb 9, 2020, and were willing to donate their blood samples. All enrolled cases were confirmed to be infected with SARS-CoV-2 by use of real-time RT-PCR (rRT-PCR) on samples from the respiratory tract. For all enrolled patients, the date of illness onset, clinical classification, RNA testing results during the hospitalization period, and the personal demographic information were obtained from the clinical records. This study was reviewed and approved by the Medical Ethical Committee of 6 Shenzhen Third People's Hospital (2020-0018). Written informed consent was obtained from each enrolled patient. The Ab, IgM antibody and IgG antibody against SARS-CoV-2 in plasma samples were For continue variables description, mean with standard deviation was used for normal distribution data and median with interquartile range (IQR) was used for non-normal distribution data. Cumulative seroconversion rates were calculated by Kaplan-Meier method. The association between antibody level and severity of illness were estimated by generalized estimating equations (GEE) model with logit link function. All statistical analysis was conducted by SAS 9.4 (SAS Institute, Cary, NC, USA). Among totally 368 COVID-19 patients admitted in the hospital (before Feb 9, 2020), 173 of them (47%) were enrolled in the study ( Table 1 ). The median age of the studied patients was 48 years (IQR, 35-61) and 51.4% were females. There were 116 (67%) patients had clear epidemiological travel/residence history in Wuhan. Among them, 32 (18.5%) were in critical illness condition. By February 19, a total of 62 patients (35.8%) were recovered and discharged from hospital and 2 (1.1%) patients died with underlying chronic disease. A total of 535 plasma samples collected during the hospitalization period of the 173 patients were tested for antibodies against SARS-CoV-2. The seroconversion rate for Ab, IgM and IgG was 93.1% (161/173), 82.7% (143/173) and 64.7% (112/173), respectively (Table 1) . Twelve patients who remained seronegative for Ab testing possibly due to that their samples involved were all collected at the early stage of illness (10 earlier than day-10, the other two on day-11 and-13 after onset). The cumulative seroconversion curve showed that the rate for Ab and IgM reached 100% around 1-month after onset. The seroconversion was sequential appeared for Ab, IgM and then IgG ( Figure 1A ). The median time to Ab, IgM and IgG seroconversion was 11-, 12-and 14-day, separately. One of two patients tested on the onset day was seropositive. Overall, the seroconversion of Ab was significantly quicker than that of IgM (p = 0.012) and IgG (p<0.001), that possibly attributed to the double-antigen sandwich form of the assay used which usually show much higher sensitivity than capture assay (IgM) and indirect assay (IgG). Moreover, all isotypes of viral specific antibodies, including IgM, IgA and IgG, can be detected by double-sandwich assay, which may also contribute to the superior performance of Ab test. In comparisons of seroconversion rates of antibodies between critical and non-critical patients, none of the three markers showed significant difference (data not shown). 8 In the early phase of illness within 7-day since onset, the RNA test had the highest sensitivity of 66.7%, whereas the antibody assays only presented a positive rate of 38.3% (Table 2) . However, the sensitivity of Ab overtook that of RNA test since day 8 after onset and reached over 90% across day 12 after onset ( Figure 1B) (Table S1 ). Whatever, combined use of the tests of RNA and Ab improved markedly the sensitivities of pathogenic-diagnosis for COVID-19 patients in different phases ( Table 2 ). To investigate the dynamics of antibody level according disease course, the antibody levels were expressed using the relative binding signals compared to the cutoff value of each assay (S/CO). The longitudinal changes of antibody and RNA in 9 representative patients, including 6 in non-critical group (Figure 2A week after onset and continuously elevated during the next 2 weeks ( Figure 3A ). Further analyses suggested that there was no significant difference on the average S/CO value of Ab tests between critical and non-critical patients before day 12 after onset ( Figure 3B ). However, critical patients showed significantly higher Ab S/CO values than non-critical cases in about 2-week after onset (p=0.02) and this association was not significant in either IgM or IgG tests (data not shown). For further exploration, we determined the relative Ab titer of these samples (expressed as relative optical density, rOD) by serial dilution measurements of each sample. The quantitative data of Ab titers also revealed a significant difference (p=0.004) between patients in critical and non-critical groups ( Figure 3C ). Multivariate longitudinal GEE analyses suggested that age (β=0.139, p <0.001), gender (β=1.415, p=0.006) and Ab titer (β=0.336, p=0.006) were the independent factors strongly associated with the clinical classification based on the severity (Table S2) . The present data demonstrated that typical antibody responses to acute viral infection are wildly induced in COVID-19 patients. To be expected, the total antibody was first detected, followed by IgM and IgG. The seroconversion rate and the antibody levels increased rapidly during the first two weeks, the cumulative seropositive rate reached 50% on the 11 th -day and 100% on the 39 th -day. The seroconversion time of Ab, IgM and IgG antibodies appeared consequently (p<0.05) with a median seroconversion day of 11, 12 and 14, respectively. Due to the lack of blood samples collected from patients in the later stage of illness, how long the antibodies could last remain unknown. Our results demonstrated an excellent sensitivity of Ab test in detections of patient's samples since 1-week after onset. Notably, even in the early stages of the illness within 1-week, some patients with undetectable RNA could be screened out through Ab testing. Combining RNA and antibody tests significantly raised the sensitivity for detecting patients (p<0.001). These findings indicate that serological test be an important supplement to RNA detection during the illness course. Up to date, the confirm diagnosis of SARS-CoV-2 infection entirely depend on the viral RNA testing. Even though with high analytical sensitivity, the real-world performance of RNA test is unsatisfied. Many suspected patients had to be tested for several days with multiple samples before confirm diagnosis were made, and during the waiting time they might have not enough priority to receive relevant treatments and quarantine managements. 3 These problems make the timely diagnosis of SARS-CoV-2 infection one of the bottlenecks for adapting relevant actions to limit the damage of current outbreak. Our study provided robust evidences that: 1) the acute antibody response in SARS-CoV-2 infection patient is very similar to many other acute viral infections; 2) the serological testing can be a powerful approach in achieving timely diagnosis; and 3) the total antibody is more sensitive than IgM and IgG for detecting SARS-CoV-2 infection. Thus, the antibody testing might play vital roles in the following settings: 1) for the suspected patients under the initial visit or clinically diagnosed patients has not been confirmed by RNA testing, the positive result of antibody increases the confidence to make a COVID-19 diagnosis; 2) for healthy close contact who is in the quarantine period, he/she should be deemed as a probable carriers if antibody positive, then the RNA should be tested more frequently and the close contacts of him/her should be observed; 3) for the RNA confirmed patient, seropositive indicates that the specific antibodies had been induced. Besides, epidemiological studies could be conducted using immunoassays. Additional, it can play an important role in searching potential animal hosts for SARS-CoV-2 using Ab-ELISA because the double-sandwich method makes it free from species restriction. It has been less than three months since the SARS-CoV-2 first invaded humans, and the prevalence of antibody against SARS-CoV-2 is nearly zero. Therefore, at least during the current outbreak which is likely to continue to May or June 2020, seropositive individuals could be a probably preceding infector. During this short-period, the total antibody could be considered as a recent infection marker similar as IgM. As the higher sensitivity than IgM and IgG, the Ab detection should be given high priority to be implemented in current clinical and public health 11 practice. If, unfortunately, SARS-CoV-2 become a common respiratory transmission pathogen lasting in humans, like influenzas or other less-pathogenic coronaviruses, rather than be completely eradicated as SARS-CoV-1, the serological diagnosis of acute SARS-CoV-2 infection will more depend on the detection of IgM in post-epidemic areas in the subsequent epidemic seasons. The Ab and IgG could be used to understand the epidemiology of SARS-CoV-2 infection and to assist in determining the level of humoral immunity in patients. Even then, the Ab will be a more sensitive marker for sentinel monitoring of imported cases in naive community. In addition to the diagnosis value, our study revealed a strong positive correlation between clinical severity and Ab titer since 2-week after illness onset, for the first time in COVID-19 patients. The results suggested that a high Ab titer may be considered as a risk factor of critical illness, independently from older age, male gender and comorbidities (Table S2) . Although it was still unclear how the causal relation between hormonal response and illness severity, the results raise a possible usage of the high Ab titer as a surrogate marker for worse clinical prognosis. Furthermore, it might be an evidence for the possibility of antibodydependent disease enhancement effects, which was commonly found in SARS-CoV-1 patients. 11-13 Whatsoever, our finding suggested that the clinical meanings of the level of antibody against SARS-CoV-2 during the acute phase of infection warrant further study. It should be noted that there were some limitations of this study. First, for most of RNA tests of the patients were based on upper respiratory tract specimens, the positive rate may be higher in detection using lower respiratory tract specimens, such as bronchoalveolar lavage fluid and deep tracheal aspirates, may yield higher sensitivity for RNA tests. Second, all the patients enrolled in this study were based on the positive findings of RT-PCR using respiratory samples. Untypical patients with lower respiratory viral load might be missed. Besides, the performance of RT-PCR depends on many factors such as the quality and consistency of the PCR assays being used, the skill of sample collection, and the sample types. Also, RT-PCR cannot distinguish the viable replicating virus and simply residual RNA, 12 which may affect the results. Third, we cannot evaluate the persistence of antibodies because samples were collected during the acute phase of patients. Fourth, although it had shown good specificity in healthy people, the cross-reactivity of the assay to other coronaviruses should be further assessed. In conclusion, the findings demonstrate that antibody tests have important diagnosis value in addition to RNA tests. These findings provide strong evidence for the routine application of serological antibody assays in the diagnosis and clinical management of COVID-19 patients. WHO. Coronavirus disease (COVID-2019) situation reports Clinical characteristics of 2019 novel coronavirus infection in China Clinical diagnosis of 8274 samples with 2019-novel coronavirus in Wuhan Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study Clinical features of patients infected with 2019 novel coronavirus in Wuhan Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. JAMA 2020 Characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (NCP). medRxiv 2020 Evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-nCoV infections A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster SARS-CoV-2 viral load in upper respiratory specimens of infected patients Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study Anti-spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection * Two patients missed IgM tests due to inadequate plasma samples. # One patient missed IgG tests due to inadequate plasma samples. $ There were 7, 11 and 35 patients had not been performed RNA testing during the 1-7 onset day, 8-14 onset day and 15-39 onset day, respectively. 10-12, 13-15, 16-18, 19-21, 22-39 since illness onset were pooled for analysis. Four parameter logistic (4PL) fitting curves were used to show the rising trend of antibodies We declare no competing interests.