key: cord-0926819-na8odvj7 authors: Zhang, Jin; Liu, Jianhua; Li, Na; Liu, Yong; Ye, Rui; Qin, Xiaosong; Zheng, Rui title: Serological detection of 2019-nCoV respond to the epidemic: A useful complement to nucleic acid testing date: 2020-03-06 journal: nan DOI: 10.1101/2020.03.04.20030916 sha: 4380f0251c595d6cb551e643198d2dacd3c6746c doc_id: 926819 cord_uid: na8odvj7 Corona Virus Disease 2019 (COVID-19) has spread rapidly to more than 70 countries and regions overseas and over 80000 cases have been infected, resulting in more than three thousand deaths. Rapid diagnosis of patients remains a bottleneck in containing the progress of the epidemic. We used automated chemiluminescent immunoassay to detect serum IgM and IgG antibodies to 2019-nCoV of 736 subjects. COVID-19 patients were becoming reactive(positive) for specific antibodies from 7-12 days after the onset of morbidity. Specific IgM and IgG increased with the progression of the disease. The areas under the ROC curves of IgM and IgG were 0.988 and 1.000, respectively. Specific antibody detection has good sensitivity and specificity. Detection of specific antibodies in patients with fever can be a good distinction between COVID-19 and other diseases, so as to be a complement to nucleic acid diagnosis to early diagnosis of suspected cases. Coronaviruses (CoVs) are enveloped single-stranded positive-sense RNA viruses, which are widely distributed in humans and other mammals. Coronaviruses usually cause respiratory, digestive and nervous system diseases in humans and animals (1) . In the past 20 years, coronavirus has caused two global epidemics of severe respiratory infectious diseases, one of which was severe acute respiratory syndrome (SARS) (2, 3) the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint swab (5-9). However, in the actual diagnosis and treatment, the sensitivity of nucleic acid detection was not ideal enough. Only 30-50% of the confirmed COVID-19 cases had positive results, moreover, in some confirmed case, nucleic acid testing often took four or more tests to get a positive result. It is necessary to use a fast and convenient method to realize the rapid diagnosis of 2019-nCoV infection. After the virus infects the organism, the immune system carries on the immune defense to the virus and produces the specific antibody. In the laboratory diagnosis of infectious diseases, the detection of specific antibodies to pathogens is a sensitive method for fast diagnosis. However, how the 2019-nCoV antibody produced and changed during COVID-19 progression is still unclear. In this study, we used automated chemiluminescent immunoassay to detect serum IgM and IgG antibodies to 2019-nCoV, to understand the process of antibody production in disease progression, and to evaluate the value of antibody detection in the laboratory diagnosis of COVID-19. The study was conducted in accordance with the International Coordinating Council for Clinical Trials and the Helsinki Declaration, and was approved by the Hospital Ethics Review Committee (Ethics No 2020PS038K), and the patient's informed consent was exempted. All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is Another 222 outpatients with other diseases in the same period, 63 medical staffs worked for fever clinic and 223 healthy physical examinees in 2018 were collected and were named other disease group, medical staff group and health control group, respectively. According to the unified form, two residents collected clinical data from medical records separately. Blood sampling Fasting venous blood (5ml) was collected from all the subjects and put into the yellow head vacuum tube containing separation gel. After centrifugation, the serum samples were stored at-20 ℃. The CT value of 2019-nCoV nucleic acid test results should be interpreted according All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint to the recommendations of the manufacturer's instructions, and the suspicious results should be notified for clinical re-sampling and re-examination. In order to be diagnosed as positive in laboratory test results, it is necessary to meet the standard that 2019-nCoV ORF1ab and N gene of same sample shows at least one target specific RT-PCR test result is positive. The diagnosis was made according to the "Diagnosis and Treatment plan of Quantitative variables were expressed as median (P99). The normality of variables All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint was tested using Kolmogorov-Smirnov test. and LDS-t-test for comparison among groups. A receiver operating characteristic curve (ROC) was plotted to evaluate the diagnostic performance and correlations determined by Spearman's rank correlation. Z test was used to compare AUC between two groups. All tests were two sided and P values < 0.05 were considered statistically significant. Of the 3 cases of confirmed case, 2 were male and 1 was female. The age ranged from 39 to 57 years. 2 patients had diabetes and hypertension respectively. 1 was a common case, 2 were severe cases. 1 case had history of Wuhan contact and the other 2 cases had no clear epidemiological history (Table 1 ). The main laboratory findings of COVID-19 patients were normal or slightly low white blood cells and lymphocytes, elevated inflammatory indicators such as interleukin-6, procalcitonin, C-reactive protein, serum amyloid A, erythrocyte sedimentation rate; and normal myocardial markers ( Table 2 ). the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint showed that the level of anti-2019-nCoV IgG was continue higher than that of IgM, and the other two cases showed that the level of anti-2019-nCoV IgM increased more than that of IgG from 2 weeks of morbidity (Table 3 ). In non-COVID-19, other disease, medical staff and health control groups, there were a few cases reactive for 2019-nCoV IgM and IgG, all the cases were single reactive for IgM or IgG. The sensitivities of IgM and IgG were 100%, as for specificities of IgM and IgG were all over 97% (Table 4 ). Of 225 non-COVID-19 cases, 2 cases were detectable for influenza A RNA and 2 cases were detectable for influenza B RNA, respectively, 4 cases were detectable for adenovirus DNA, 17 cases were detectable for mycoplasma pneumonia DNA (Table 4 ). We also compared the anti-2019-nCoV antibodies values distributions in different groups. The anti-2019-nCoV IgM levels in non-COVID-19 was higher than that of healthy control group, the difference was statistically significant (Table 4 ; The area under the curve was 0.988 and 1.000, and the best cut-off value was 10.14 and 15.99, respectively ( Figure 3 ). With China's growing surveillance network and laboratory capacity, the All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint outbreak was identified within a few weeks and the viral genome sequence was announced (10), effectively promoting in vitro diagnostic tests. At present, the main diagnostic method is to detect 2019-nCoV nucleic acid by real-time quantitative fluorescent PCR. In the early stage of this epidemic, due to the insufficient production of nucleic acid detection kits and the high requirement of technical norms for nucleic acid detection, the application of nucleic acid testing as a diagnostic standard was limited, When the body's immune system produces antibodies, the clinical signs and symptoms is obvious, the virus is likely to decline without being detected. All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is Recently, a study of 138 patients showed a high proportion (41%) of suspected nosocomial infection (11) , and it is also noteworthy that the presence of asymptomatic patients with latent mild pneumonia may be an important source of infection for outbreak transmission (12) . Therefore, it remains critical to apply a fast and convenient detection method to distinguish and trace suspicious case or contacts as early as possible in order to prevent super-transmission events. Antibodies are the products of humoral immune response after infection with viruses. As a new infectious disease, while the detection of nucleic acid cannot be used widely, specific antibodies to 2019-nCoV can be used to determine whether the patient has been recently infected with 2019-nCoV or not. It has been reported that serum samples from 5 patients were detected by self-made 2019-nCoV IgG and IgM ELISA kits, and the antigen could cover 92% of the 2019-nCoV NP amino acids (13) . Generally speaking, the immune response of pathogenic microorganisms is usually stimulated by the rise of IgM after infection, IgG usually appears 1-2 weeks after IgM, and has been rising and maintaining high levels in the body for a long time. Because COVID-19 is a new infectious disease and the immunological test reagent has just been developed, there is still no report on how IgM and IgG antibodies were produced and developed after 2019-nCoV infection. In our study, we found that specific antibodies reactive to 2019-nCoV appeared from 7-12days after the onset of morbidity in all 3 patients. Unlike previous experience that IgG usually appears 1-2 weeks after IgM, the presence of anti-2019-nCoV IgM antibodies in COVID-19 cases was followed by the presence of All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is 2019-nCoV is highly infectious and the population is generally susceptible to 2019-nCoV.The most common symptoms after infection include fever, fatigue, dry cough, and muscle pain, with expiratory dyspnoea occurring in more than half of patients (14) . Severe cases are prone to rapidly progress to acute respiratory distress syndrome, septic shock, high risk of admission to intensive care units, and even death. Therefore, how to closely observe the condition after morbidity and find severe cases as soon as possible is the key to reduce the mortality of critically ill patients. According to our findings, it seems that the time and speed of production of specific anti-2019-nCoV IgM antibodies correlate with disease severity. But because the number of cases is so small, more research is needed to confirm it. In addition to COVID-19, the fever patient of non-COVID-19, other disease, All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint medical staffs and healthy controls were also studied. Non-COVID-19 group included several other respiratory viruses such as influenza A, B and adenovirus infection cases, these cases were negative for anti-2019-nCoV specific antibody detection, indicating that the antibody detection has a good ability to resist interference and differential Considering that COVID-19 has broken out in many countries around the world, more than 80000 people have been diagnosed and the number is growing rapidly, the main problem at present is the need for highly sensitive tests to screen the suspected cases and to prevent missed diagnosis by nucleic acid tests, lower false positive rates for antibody testing are acceptable. In the meantime, for patients with morbidity for a week or more, simultaneous positive of anti-2019-nCoV IgM and IgG will be helpful to improve the specificity. Compared with nucleic acid test, which requires respiratory tract samples and complex testing procedures, the operation requirement of serum antibody detection in clinical laboratory is lower than that of nucleic acid detection, which can be detected quickly (30min) and in large quantities, and can be completed in common P2 Biosafety Laboratory. When the morbidity is more than a week, nucleic acid detection is not convenient, serological dynamic monitoring can be carried out, once positive, it All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint is strongly recommended to use nucleic acid diagnosis immediately. The disadvantage of nucleic acid detection is the existence of relative high false negative rate, and serological antibody detection has the advantage of high sensitivity, so the combination of the two will be a good diagnostic means. It can be inferred that after the future epidemic situation has been controlled to a certain extent, as a convenient method, antibody detection is still necessary to make differential diagnosis of other respiratory pathogens infection. It must be emphasized that independent results of specific antibodies testing should not be used as a diagnostic criteria, especially when the epidemiological history is unclear, and must be combined with the patient's morbidity time and clinical signs.It must be emphasized that independent results of specific antibodies testing should not be used as a diagnostic basis, especially when the epidemiological history is unclear, and must be combined with the patient's morbidity time and clinical signs. To our knowledge, little has been reported about the specific antibody production process in the course of COVID-19 disease, and little has been reported about the different situation of antibodies in fever non-COVID-19 population, other diseases, special contact population such as medical staff and healthy population. This study provides data on the regularity of antibody production in the course of COVID-19, and provides some understanding of the basic data of specific antibodies in different populations. The results of this study help to provide evidence for rapid screening of suspected cases through the serological testing to curb the rapid progress of the epidemic globally. Just on the day of this manuscript was submitted, the China All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is Considering that Liaoning Province, where this study was conducted, is a low epidemic area, the possibility of asymptomatic infection would be very small. In this paper, we studied the producing process of specific antibody in patients with COVID-19, compared and evaluated the diagnostic value of antibody in different populations, which is beneficial for doctors to use in the process of diagnosis and treatment. As a useful complement to nucleic acid detection，the detection of specific anti-2019-nCoV antibodies will be able to draw a more comprehensive, rapid and accurate diagnosis to COVID-19, so as to effectively distinct between COVID and non-COVID-19 patients and curb the rapid spread of 2019-nCoV in the global epidemic period. All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint We declare no competing interests. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint Table2. Laboratory findings of 3 patients with COVID-19 Normal range case1 case2 case3 white blood cell count,x10 9 /L 3.5-9.5 5.0 6.3 4.0 neutrophil count,x10 9 /L 1.9-7.2 3.7 4.1 3.4 Lymphocyte count,x10 9 /L 1. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint Table 3 . Anti-2019-nCoV antibody production Emerging coronaviruses: Genome structure, replication, and pathogenesis A novel coronavirus associated with severe acute respiratory syndrome Characterization of a novel coronavirus associated with severe acute respiratory syndrome Genomic and protein structure modelling analysis depicts the origin and infectivity of 2019-nCoV Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster A pneumonia outbreak associated with a new coronavirus of probable bat origin All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is China National Health Commission. Diagnosis and Treatment plan of Corona Virus Disease No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint (which was not peer-reviewed) is All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint Table4 Anti-2019-nCoV antibody detection in different groups non-COVID-19 other Disease medical staff health control number 225 222 63 the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint Figure 1 . Dynamics of antibody production in 3 patients All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.04.20030916 doi: medRxiv preprint