key: cord-0892420-8mx5kowl authors: Gao, Yong; Yuan, Yi; Li, Tuan Tuan; Wang, Wu Xiao; Li, Yong Xiu; Li, Ang; Han, Feng Ming title: Evaluation the auxiliary diagnosis value of antibodies assays for detection of novel coronavirus (SARS-Cov-2) causing an outbreak of pneumonia (COVID-19) date: 2020-03-30 journal: nan DOI: 10.1101/2020.03.26.20042044 sha: 0dbd5520b71aae8af4f31fee2d4a7db593a138e2 doc_id: 892420 cord_uid: 8mx5kowl Background: The spread of an novel coronavirus (SARS-CoV-2, previously named 2019-nCoV) has already taken on pandemic proportions, affecting over 100 countries in a matter of weeks. Elucidating the diagnostic value of different methods, especially the auxiliary diagnosis value of antibodies assays for SARS-CoV-2 infection is helpful for improving the sensitivities of pathogenic-diagnosis, providing timely treatment, and differentiating the infected cases from the healthy, thus preventing further epidemics. Methods: Medical records from 38 patients with confirmed SARS-CoV-2 infection in the Second People's Hospital of Fuyang from January 22, 2020 to February 28, 2020 were collected and retrospectively analyzed. Specimens including throat swabs, sputum and serum were collected during the hospitalization period, viral RNAs and serum IgM-IgG antibodies to SARS-CoV-2 were measured respectively. The detectability of different methods as well as the auxiliary diagnosis value of antibodies test for SARS-CoV-2 infection were analyzed. Results: Among 38 patients, the total seropositive rate for IgM and IgG was 50.0% and 92.1%, respectively. Two patients remained seronegative throughout the course of illness. In the early phase of illness, the RNA test for sputum specimens possessed the highest detectability(92.3%), followed by the the RNA test for throat swabs (69.2%), and the antibodies assays presented lower positive rates(IgM, 23.0%, IgG, 53.8%). While, the sensitivity of antibodies assays overtook that of RNA test since day 8 after onset (IgM, 50.0%; IgG, 87.5%). Of note, the positive rate of throat swabs was only 13.0% for cases in later phase(≥15 d.a.o), and the sensitivities of IgM and IgG rose to 52.2% and 91.3%, respectively. Combined use of antibodies assay and qRT-PCR at the same time was able to improve the sensitivities of pathogenic-diagnosis, especially for the throat swabs group at the later stage of illness. Moreover, most of these cases with undetectable viral RNA in throat swabs specimens at the early stage of illness were able to be IgM/IgG seropositive after 7 days. Conclusions: The antibodies detection against SARS-CoV-2 offers vital clinical information for physicians, and could be used as an effective supplementary indicator for suspected cases of negative viral nucleic acid detection or in conjunction with nucleic acid detection in the diagnosis of suspected cases. While, the sensitivity of antibodies assays overtook that of RNA test since day 8 after onset (IgM, 50.0%; IgG, 87.5%). Of note, the positive rate of throat swabs was only 13 .0% for cases in later phase(≥15 d.a.o), and the sensitivities of IgM and IgG rose to 52.2% and 91.3%, respectively. Combined use of antibodies assay and qRT-PCR at the same time was able to improve the sensitivities of pathogenic-diagnosis, especially for the throat swabs group at the later stage of illness. Moreover, most of these cases with undetectable viral RNA in throat swabs specimens at the early stage of illness were able to be IgM/IgG seropositive after 7 days. The antibodies detection against SARS-CoV-2 offers vital clinical information for physicians, and could be used as an effective supplementary indicator . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 30, 2020. . https://doi.org/10.1101/2020.03.26.20042044 doi: medRxiv preprint for suspected cases of negative viral nucleic acid detection or in conjunction with nucleic acid detection in the diagnosis of suspected cases. . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The outbreak of coronavirus disease 2019 , caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, continues to spread worldwide and has become a global threat. As of March 21, 2020, it has reached more than 115 countries, with 266073 cases and 11184 deaths (1) (2) . The World Health Organization has declared the COVID-19 outbreak a pandemic and rates the global risk assessment as very high.A global response to prepare health systems worldwide is imperative(3).Timely and accurate diagnosis of suspected SARS-CoV-2 infection, and to isolate and care for these patients early are of great importance for interrupting human-to-human transmission, and limiting further spread of the virus. Common laboratory abnormalities in confirmed COVID-19 cases include decreased white blood cells, lymphocytes, and platelet counts, and an increased LDH, CK, and CRP levels (4) (5) . And the reported common clinical symptoms include fever, cough, myalgia or fatigue (6) . However, these abnormalities and symptoms are not unique features of COVID-19 since these might be similar to that of other virus infected disease. Meanwhile, some infected patients are asymptomatic but can also become a source of infection, which makes early diagnosis essential. The use of quantitative real-time PCR (qRT-PCR) assays for the detection of the viral nucleic acid has become the primary and crucial diagnostic approach for identification of SARS-CoV-2 infection, while it still has some limitations in clinical practice (7) (8) (9) . The RNA-based diagnostic tests only give a positive result when the virus is still present. The tests could not identify people who has been infected in the past, recovered, and cleared the virus from their bodies. In addition, negligible false-negative risk brought by PCR test were reported and the positive rates varied for different specimens in COVID-19 patients. A number of cases that were epidemiologically linked to SARS-CoV-2 exposure and with typical lung CT images still remained RNA negative in their respiratory tract specimens(10-12). IgG/IgM antibodies test, a serological test method, has been added as a diagnostic criteria in China's updated version of the diagnosis and treatment guidelines for COVID-19 (3rd, March).It will help to trace in a much more is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 30, 2020. . https://doi.org/10.1101/2020.03.26.20042044 doi: medRxiv preprint population-based way whether a person has been infected in the past since the body could retain antibodies against virus that it has already overcome.While such assays still need to be carefully validated to be sure they could react only to antibodies against SARS-CoV-2. The similarity between SARS-CoV-2 and the other viruses might lead to cross-reactivity. Also here were still false positive and false negative results for IgG/IgM antibodies test (13) (14) (15) . Herein, there is an urgent need for elucidating the diagnostic accuracy of different specimens and methods to improve the positive rates, so as to prevent virus transmission and to assure timely treatment of patients. Respiratory specimens including throat swabs and sputum were collected, and then the throat swabs were placed into a sterile test tube with 1 mL sterile saline, the sputum samples were added equal volume of acetylcysteine and shaken at room temperature for 30 min to be fully liquefied. Next, total RNA was extracted using a viral nucleic acid isolation kit (Jiangsu Bioperfectus Technologies Company, Ltd.), is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 30, 2020. Serum IgG and IgM antibodies against SARS-CoV-2 were tested by using a GICA kits according to the manufacturer's protocol (Innovita Biological Technology Co., Ltd.). Briefly, for each test, 10μL of serum sample and 80μL of sample diluent were added onto the pad of the test strip. Then the strip was placed flat at room temperature for 15min to react and then result could be judged according to the color of the tested and control lines. ① Both the detection band and the control band turn red, the sample will be interpreted as positive.② If the control band turns red while the detection band does not, it will be interpreted as negative. ③ If neither band was colored, the test reagents will be assumed to be not working and required confirmation by retesting. All analyses were performed using SPSS 19.0. Continuous variable data were in the median (Interquartile range, IQR), categorical variables were expressed as frequencies (percentages), chi-square test with Yates's correction or Fisher's exact test was used for comparison between groups. P<0.05 was considered to be . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 30, 2020. . https://doi.org/10.1101/2020.03.26.20042044 doi: medRxiv preprint statistically significant. Medical records from 38 COVID-19 patients were collected and retrospectively analyzed, the median age was 40.5 years (IQR, 31.0-49.5years) and 55.3% were males. Of these patients, 3 cases were in severe or critical conditions, and the rest were mild cases. The median number of specimens collected from each patient was 8. We analyzed the detectability of RNA test and antibodies assays according to the time course since illness onset in the cohort. As the results shown in is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Based on the above mentioned findings, we aimed to evaluate the auxiliary diagnosis value of antibodies assays for suspected cases of negative nucleic acid detection. Firstly, the detectability of antibodies assays in patients with undetectable viral RNA in their respiratory tract specimens were analyzed. As the results shown in Table 2 and Table 3 , combined use of the tests of RNA and antibodies assay at the same time was able to improve the sensitivities of pathogenic-diagnosis for SARS-CoV-2 infection, especially for the throat swabs group at the later stage of illness.Then, we further analyzed the antibodies test data of cases with undetectable viral RNA in their throat swabs specimens at the early stage of illness. Most of them were shown to be IgM/IgG seropositive after 7 days of negative nucleic acid test results (IgM + 47.1%, IgG + 91.1%), suggesting an auxiliary diagnosis value of antibodies assays. The spread of a novel coronavirus (SARS-CoV-2) has recently been identified in patients with acute respiratory disease.It is the third highly pathogenic and transmissible coronavirus after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in humans (16) . Effective diagnosis that identify SARS-CoV-2 infection in people are of great importance for public health efforts, not just for individuals' health concerns. Patients' clinical manifestations mainly included fever, cough, dyspnea, myalgia, fatigue, and radiographic evidence of pneumonia. Diagnosis could be based on clinical history, laboratory and CT images, but confirmation primarily relies on nucleic acid detection. Yet many novel coronavirus pneumonia patients can not be diagnosed due to negative nucleic acid test. For example, throat swab is commonly used for nucleic acid test, while the viral loads in upper respiratory tract samples are usually much lower than that in lower respiratory tract samples in COVID-19 cases, and the viral loads of patients varies in different stage of illness (17) . Consequently, these problems lead to an urgent need for clinical auxiliary diagnosis, so as to improve the positive detective rates and to provide timely treatment is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 30, 2020. . https://doi.org/10.1101/2020.03.26.20042044 doi: medRxiv preprint and preventive quarantine. The human immune system can produce specific IgM and IgG antibodies against virus infection. IgM is the earliest antibody that appears upon first immune response. Serological presence of IgM indicates a recent infection and could be used as auxiliary diagonosis of early infection. IgG is produced later and lasts long, which can be used as an indicator of previous or secondary infection (18) (19) . In this study, the positive rate of throat swabs was only 13.0% for cases in later phase( ≥ 15 d.a.o), and the sensitivities of IgM and IgG were 52.2% and 91.3%, respectively. Combining use of antibodies assay at the same time could be able to greatly improve the sensitivities of pathogenic-diagnosis for SARS-CoV-2 infection, especially for the throat swabs group during the later stage of illness.Moreover, through further analysis of the antibodies test data of cases with undetectable viral RNA in throat swabs specimens at the early stage of illness, we suggest that most of these cases could be IgM/IgG seropositive after 7 days of negative nucleic acid test results, indicating an auxiliary diagnosis value of antibodies assays. It should be noted that there were still false positive and false negative results for antibodies assay. When IgM and IgG levels are below the detection limit, the test results would be negative. Furthermore, IgM antibodies would gradually decrease and disappear after 2 weeks, so that the IgM level might be below its peak and not detectable in some cases.Meanwhile, the difference in individual immune response might result in the false negative results in suspected cases (19) (20) . Patients who do not produce antibodies, or who produce antibodies relatively late, might have a relapse once their immunity is reduced. In conclusion, the test of IgM and IgG antibodies against SARS-CoV-2 provides important immunological evidence for physicians, and could be used as an effective is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 30, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted March 30, 2020. . https://doi.org/10.1101/2020.03.26.20042044 doi: medRxiv preprint Coronavirus disease (COVID-2019) situation reports A Novel Coronavirus from Patients with Pneumonia in China Understanding of COVID-19 based on current evidence Clinical and Biochemical Indexes from 2019-nCoV infected patients linked to viral loads and lung injury Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia Evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-nCoV infections A pneumonia outbreak associated with a new coronavirus of probable bat origin Application and optimization of RT-PCR in (SARS-Cov-2) infected pneumonia Recent advances in the detection of respiratory virus infection in humans High incidence of asymptomatic SARS-CoV-2 infection Long-Term Persistence of IgG Antibodies in SARS-CoV Infected Healthcare Workers Antibody responses to SARS-CoV-2 in COVID-19 patients: the perspective application of serological tests in clinical practice Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia Novel Coronavirus can be detected in urine, blood, anal swabs and oropharyngeal swabs samples A serological assay to detect SARS-CoV-2 seroconversion in humans Evaluation of Enzyme-Linked Immunoassay and Colloidal Gold-Immunochromatographic Assay Kit for Detection of Novel Coronavirus (SARS-Cov-2) Causing an Outbreak of Pneumonia It is made available under a perpetuity.is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The authors declare no financial or commercial conflict of interest. This work was supported by "The Science and Technology Bureau of Fuyang (202004a07020009)". The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted March 30, 2020. . https://doi.org/10.1101/2020.03.26.20042044 doi: medRxiv preprint