key: cord-0981340-rbnfh89u authors: Xiang, Jie; Yan, Mingzhe; Li, Hongze; Liu, Ting; Lin, Chenyao; Huang, Shuang; Shen, Changxin title: Evaluation of Enzyme-Linked Immunoassay and Colloidal Gold- Immunochromatographic Assay Kit for Detection of Novel Coronavirus (SARS-Cov-2) Causing an Outbreak of Pneumonia (COVID-19) date: 2020-03-01 journal: nan DOI: 10.1101/2020.02.27.20028787 sha: b579c3547bba2a33057373d57b7c05f37dd4cfc3 doc_id: 981340 cord_uid: rbnfh89u Abstract BACKGROUND: In December 2019, a novel coronavirus (SARS-CoV-2) infected pneumonia (COVID-19) occurred in Wuhan, China. Travel-associated cases have also been reported in other countries. The number of cases has increased rapidly but laboratory diagnosis is limited. METHODS: We collect two groups of cases diagnosed with COVID-19 for experiments. One group collected 63 samples for Enzyme-linked immunosorbent assay (ELISA) IgG and IgM antibodies. The other group collected 91 plasma samples for colloidal gold-immunochromatographic assay (GICA). RESULTS: The sensitivity of the combined ELISA IgM and ELISA IgG detection was 55/63 ( 87.3%), The sensitivity of the combined GICA IgM and GICA IgG detection was 75/91 ( 82.4%), Both methods are negative for healthy controls, specificity of 100% .There is no significant difference between the sensitivity of between ELISA and GICA (IgM+ IgG). CONCLUSIONS: ELISA and GICA for specific IgM and IgG antibodies are conventional serological assays, they are simple, fast, and safe, the results can be used for clinical reference, and the huge clinical diagnosis and treatment pressure can be greatly relieved. greatly relieved. In December 2019, a cluster of acute respiratory illness, now known as novel coronavirus-infected pneumonia (NCIP), occurred in Wuhan, Hubei Province, China novel coronavirus is a highly infectious disease, and the ongoing outbreak has been declared by WHO as a global public health emergency. A novel coronavirus of zoonotic origin (2019-nCoV) has recently been identified in patients with acute respiratory disease. This virus is genetically similar to SARS coronavirus and bat SARS-like coronaviruses. Outbreaks in health care workers indicate human-to-human transmission. Full-genome sequencing and phylogenic analysis indicated that 2019-nCoV is a distinct clade from the beta coronaviruses associated with human severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) (1) . International Committee on Taxonomy of Viruses(ICTV)is announced that 2019-nCoV is officially classified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)(4,5 Diagnosis is based on clinical history and laboratory and chest radiographic findings, but confirmation currently relies on nucleic acid-based assays. The nucleic acid-based assay has been used to confirm the diagnosis of new pneumonia, but many patients will miss the diagnosis. We have verified the two new reagents kit that are All rights reserved. No reuse allowed without permission. 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 . https://doi.org/10.1101/2020.02.27.20028787 doi: medRxiv preprint currently not on the market and have achieved good results. The following are reported as follows: This case series was approved by the institutional ethics board of Wuhan Jinyintan Hospital. All consecutive patients with confirmed COVID-19 admitted to Jinyintan Hospital from January 1 to January 28, 2020, were enrolled. Oral consent was obtained from patients. Jinyintan Hospital, located in Wuhan, Hubei Province, the endemic areas of COVID-19, is first designated hospital for COVID-19 patients in Wuhan. All patients with COVID-19 enrolled in this study were diagnosed according 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 After 10 min, the result was judged by the color of the test and control lines. If both the detection band and the control band turned red, the sample was recorded as positive. If the control band turned red but the detection band was not colored, it was recorded as negative. If neither band was colored, the test reagents were assumed to be not working. Throat swab samples, sputum samples and alveolar lavage fluid samples were All rights reserved. No reuse allowed without permission. 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 . https://doi.org/10.1101/2020.02.27.20028787 doi: medRxiv preprint collected for extracting 2019-nCoV RNA from patients suspected of having 2019-nCoV infection. After collection, the throat swabs were placed into a sterile test tube with 1 ml sterile saline, the sputum samples were added equal volume of acetylcysteine (10g/L) and shaken at room temperature for 30 min to be fully liquefied, and total RNA was extracted using the nucleic acid extraction kit (QIAamp viral RNA mini kit). In brief, 40μL 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 centrifugated at 1000 rpm/min for 5 minutes. The All analyses were performed using SPSS 22.0. Categorical variables were expressed as frequencies (percentages), and performed using the chi-square test with Yates's All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Table 3 and Table 4) qRT-PCR results qRT-PCR test of 81 samples of known COVID-19 patients, the results showed that 42 cases were positive, the sensitivity was 51.9% (42/81). Overall, there are significant differences in the three detection methods (P<0.001). There is no significant difference between the sensitivity of between ELISA (IgM+ IgG) and GICA (IgM+ IgG), P=0.411. (Table 3) All rights reserved. No reuse allowed without permission. 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 . https://doi.org/10.1101/2020.02.27.20028787 doi: medRxiv preprint At present, the sample collection for nucleic acid detection of suspected cases of COVID-19 is mostly upper respiratory tract samples (mainly throat swabs) (9). The collection of throat swabs is not standardized and it is easy to miss the diagnosis. The collection process is extremely risky for medical staff. Confirmed diagnosis is playing an important role in facilitating patient isolation, treatment and assessment of infectious activities. However, due to their limited capacity to handle an epidemic of the current scale and insufficient supply of assay kits, only a portion of suspected cases can be tested, leading to incompleteness and inaccuracy in updating new cases, as well as delayed diagnosis. A fast-performing serologic assay is acutely needed for the current and outbreak. In this study, we tested serum samples from confirmed COVID-19 patients with two assay kits and achieved a high positive rate. The sensitivity of the combined ELISA IgM and IgG detection was 55/63 (87.3%), The sensitivity of the combined GICA IgM and IgG detection was 75/91 (82.4%), and the healthy controls were negative. The case group was COVID-19 patients diagnosed by qRT-PCR. Since many patients are undergoing treatment, they are gradually recovering, the nucleic acid test result has become negative, so the positive rate of patients tested was only 51.9%. It is worth noting that the new type of coronavirus antibody of the kit is against the severe acute respiratory syndrome (SARS)-like coronavirus, not only against SARS-CoV-2, but the IgG originally infected with SARS may also be positive. But our research found, the healthy controls are all negative and the specificity is very good. ELISA and GICA for specific IgM and IgG antibodies is conventional serological assays, they can offer a high-throughput alternative, which allows for uniform tests for all suspected patients, and can facilitate more complete identification of infected cases and avoidance of unnecessary cross infection among unselected All rights reserved. No reuse allowed without permission. 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 . https://doi.org/10.1101/2020.02.27.20028787 doi: medRxiv preprint patients. They use plasma or serum as test samples, and blood samples can be collected easily, which can greatly reduce the risk of infection for medical staff. The complicated processing procedure of the sample during the laboratory test is removed, the test result can be obtained quickly, the operation is simple, the safety of medical staff can be protected, and the huge clinical diagnosis and treatment pressure can be greatly relieved. Although ELISA and GICA are simple, fast, and safe, the results can be used for clinical reference. Cases confirmation still depended on qRT-PCR, and analyze epidemiological, demographic, clinical, and radiological features and other laboratory data. There is a need to further increase the number of samples tested to prove their t of sample testing to confirm their significance. In conclusion, ELISA and GICA for specific IgM and IgG antibodies is conventional serological assays, they are simple, fast, and safe for diagnosis COVID-19. The results can be used for clinical reference, and the huge clinical diagnosis and treatment pressure can be greatly relieved. 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 . https://doi.org/10.1101/2020.02.27.20028787 doi: medRxiv preprint 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 . https://doi.org/10.1101/2020.02.27.20028787 doi: medRxiv preprint 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 . https://doi.org/10.1101/2020.02.27.20028787 doi: medRxiv preprint Table 3 Compare the sensitivity of the three methods All rights reserved. No reuse allowed without permission. 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 . https://doi.org/10.1101/2020.02.27.20028787 doi: medRxiv preprint A Novel Coronavirus from Patients with Pneumonia in China This work was supported by the Zhongnan Hospital of Wuhan University Science, Technology and Innovation Seed Fund under Grant znpy2017022. The authors have no conflict of interest.