key: cord-0809906-ofazmvjf authors: Wan, Y.; Li, Z.; Wang, K.; Li, T.; Liao, P. title: Performance verification of detecting COVID-19 specific antibody by using four chemiluminescence immunoassay systems date: 2020-05-02 journal: nan DOI: 10.1101/2020.04.27.20074849 sha: a586240e8d42c215a8a3cbd73f434eb6ea4d184f doc_id: 809906 cord_uid: ofazmvjf Background The purpose of current study is to evaluate the analytical performance of seven kits for detecting IgM/IgG antibody of corona virus (2019-nCoV) by using four chemiluminescence immunoassay systems. Methods 50 patients diagnosed with 2019-nCoV infection and 130 controls without corona virus infection from the people's hospital of Chongqing were enrolled in current retrospective study. Four chemiluminescence immunoassay systems including seven IgM/IgG antibody detection Kits for 2019-nCoV (A_IgM, A_IgG, B_IgM, B_IgG, C_IgM, C_IgG, D_Ab) were employed to detecting antibody concentration. Chi-square test,receiver operating characteristic (ROC) curve and Youden's index were demonstrated to verify the cutoff value of each detection system. Results The repeatability verification results of the A, B, C, and D system are all qualified. D-Ab performances best (92% sensitivity and 99.23% specificity), and B_IgM worse than other systems. Except for the system of A_IgM and C_IgG, the optimal diagnostic thresholds and cutoff value of other kits from recommendations are inconsistent with each other. B_IgM got the worst AUC and C_IgG had the best diagnostic accuracy. More importantly, B_IgG system have the highest false positive rate for testing patients with AIDS, tumor and pregnant. A_IgM system test showed highest false positive rates among elder over 90 years old. Conclusions Systems for CoVID-2019 IgM/IgG antibody test performance difference. Serum diagnosis kit of D-Ab is the most reliable detecting system for 2019-nCoV antibody, which can be used as an alternative method for nucleic acid testing. The corona virus pneumonia (Corona Virus Disease 2019, COVID-19) is an acute respiratory infection caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) [1] . The epidemic of the disease has not ended since the winter of 2019 and it is still raging around the world. SARS-CoV-2 is highly contagious through air, droplets and contacts. Generally, the incubation period of SARS-CoV-2 is 3-7 days, but the longest incubation period can reach 14 days [2] . It has caused more than 2,420,000 people infections and nearly 167,000 deaths worldwide until the end of April 21th. Therefore, the early diagnosis of SARS-CoV-2 infection is very crucial. Previous studies show that SARS-CoV-2 antigen stimulates the immune system to produce an immune response, and specific IgM and IgG antibodies will appear in the serum of patients after infecting [3] . The SARS-CoV-2 specific IgM and IgG antibody tests have been involved in the diagnosis criteria for suspected cases whose COVID-19 viral nucleic acid test appears false negative in recently published guideline of Novel Corona Virus Pneumonia Diagnosis and Treatment (Trial Version 7) which advocated by the National Health Committee [4] . Current popularly detection methods for SARS-CoV-2 antibodies include colloidal gold and chemiluminescence immunoassay [5] . Chemiluminescence immunoassay is All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2020. . https://doi.org/10.1101/2020.04.27.20074849 doi: medRxiv preprint a laboratory technology that combines a luminescence system with an immune response. It not only has the specificity of the immune response, but also has the high sensitivity of the luminescence reaction, and is widely used in immunoassays [6] . Our laboratory currently has four automatic chemiluminescence immunoassay systems A, B, C and D, of which the three detection systems A, B and C detect SARS-CoV-2 specific IgM and IgG antibodies respectively, and the D system detects total antibody of IgM/IgG. Current investigation intends to evaluate the repeatability, clinical sensitivity and specificity of 7 antibody detection kits for 4 detecting systems, as well as the false positive rate in special populations. The Youden's index verifies the best diagnostic threshold (Cutoff value) of each detection system to understand the analytical performance of each system detecting and ensure the detecting results. Automatic immunochemiluminescence analyzer A called detection system A (Bioscience Diagnostic Technology Co., Ltd.). Reagents include the Coronavirus (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2020. Under the condition of calibration and quality control of the detection systems, all of them are qualified and the following experiments are carried out. The cutoff value (cutoff value) is 1.0, 1.0, 1.10 AU/ml, 1.10 AU/ml, 10 AU/ml, 10 AU/ml and 1.0 in detection system of A_IgM, A_IgG, B_IgM, B_IgG, C_IgM, C_IgG, D_Ab respectively. In 50 specimens of patients infected SARS-CoV-2, one case of weak positive specimen with S/CO value within less than 3 times of cutoff value (Level1, L1) and one case with an S/CO value greater than 3 times of cutoff value (Level 2, L2) were selected. Within-run precision was conducted firstly. All detecting system analyzes their corresponding L1 and L2 specimens respectively, conducting 20 consecutive tests. All test were completed within one day, observe 20 S/CO value, judge the result, and calculate its standard deviation and coefficient of variation. The result is judged to be 100% in line, and the coefficient of variation is less than 10% is qualified. Between-run precision was conducted secondly. Detecting system analyze the corresponding L1 and L2 specimens once a day, and continuously detecting for 20 days, observation 20 times S/CO value, judge the result, and calculate All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2020. . https://doi.org/10.1101/2020.04.27.20074849 doi: medRxiv preprint 5 its standard deviation and coefficient of variation. The result is judged to be 100% in line, and the coefficient of variation is less than 15% is qualified. All statistical analyses were conducted using R software (http://www.R-project.org/). Evaluation of sensitivity with 95% CI, specificity with 95% CI and false positives in specific populations were conducted separately. Use ROC curve (R packages pROC) and Youden's index to calculate the optimal diagnostic threshold (Cutoff value) of the detection system. In order to test precision of each kit, we performed within-run and between-run detecting. As can be seen from Table 1 Overall 180 subjects were tested for COVID-19 specific serological assay. The results showed vary sensitivity and specificity among different kits. D-Ab performances best (92% sensitivity and 99.23% specificity), and B_IgM worse than others (Table2). ROC curve was depicted by using original S/CO value (Figure1). According to ROC curve, we can get optimal operating point of different kits ( Table 3) . It can be conclude that, except for the optimal operating thresholds of A_IgM and C_IgG, the optimal diagnostic thresholds of other kits and the Cutoff value from recommendations are inconsistent with each other. The results showed that the AUC of D_Ab reached 0.95 and Youden's index is 0.93 ( Table 3 ). The optimal cutoff value was 0.54, with sensitivity and specificity values of 99% and 94%, respectively. According to the optimal operating threshold, there were only 3 patients who had a negative result and two controls had a positive result. Totally, B_IgM got the worst All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2020. . https://doi.org/10.1101/2020.04.27.20074849 doi: medRxiv preprint 6 AUC and C_IgG had the best diagnostic accuracy. Considering endogenous and exogenous factors exist in the process of antibody assay, subgroups of controls including patients with AIDS (Acquired Immune Deficiency Syndrome), tumor, pregnant and elders more than 90 years old were involved in current analysis. Each system has false positive results in the selected subgroup of controls ( Table 4) . It is worth noting that B_IgG system have the highest false positive rate for testing patients with AIDS, tumor and pregnant. A_IgM system test showed the highest false positive rates among elder over 90 years old. infected human beings. Its nucleocapsid protein (NP) stimulates human immune system to cause chemical reactions. Specific IgM antibodies emergence at the 7th day of infecting, and appear at peaks after 28 days. Specific IgG antibody emerged around the 10th day of infecting, and reached peaks after 49 days, which can maintain at a long time in the blood. The median time for total plasma antibodies appear at the 12th day after infecting [7, 8] . In current investigation, the average time of serum collection in all subjects at 13 days after diagnosis, therefore it is considered that specific IgM and IgG antibodies should already exist in the specimen. Manufacturers domestically produce antibody detection reagents which are used in the clinical laboratory. Previous investigation has shown that the clinical specificity All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2020. And the clinical specificity and sensitivity of 2019-nCoV IgG antibody are 92.4% and 96.1% [6] . Therefore, the false negative and false positive results will appear in the detection, which will cause confusion to clinical judgment. So the laboratory needs to pay close attention to the performance indicators of the reagents used. Seven detection kits from four chemiluminescence systems were used in current study. According to guideline of WS/T 494-2017, the sensitivity and specificity of qualitative items for different occasions are also regulated. In the using of preliminary screening tests, the sensitivity should be greater than 95%. In the occasion of diagnose, both of the sensitivity and specificity should be greater than 95%. In a confirmed diagnostic test, the specificity should be greater than 98% [9] . According to the result of current study, the clinical sensitivity and specificity from all detecting systems does not meet the requirements of screening, diagnosis and confirm diagnosis experiments. Therefore, all detection systems cannot be used independently for the diagnosis and of SARS-CoV-2 infections, and need to be used together with nucleic acids test and clinical symptoms considering. Regarding the confounding factors influence detection results, we divided controls for subgroups which includes patients with AIDS, tumor, pregnant and older people over 90 years old [5, [11] [12] . The results of current investigation show that B_IgM has the All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2020. B_IgG has the lowest specificity which indicated higher false positives and prone to occur for special patients, such as AIDS, solid tumor, pregnant and the elderly, etc. [13] The reason of false positives may be due to some interfering substances (such as rheumatoid factor, homologous to the kit antibodies, etc.) present in the specimen. Simultaneously, according to the area under the ROC curve of each detecting system, it is found that the diagnostic accuracy of B_IgM and B_IgG also demonstrated worst, and the diagnostic accuracy of the other systems is better. In addition, according to ROC curve and Youden's index, the best diagnostic thresholds exist in A-IgM and C-IgG, and others are inconsistent with the manufacturer's declaration. The optimal threshold of A_IgG, B_IgM, C_IgM, D_Ab are less than the Cutoff value indicating more false positive results. The optimal threshold of C_IgM is greater than the Cutoff value, indicating more false negative cases. Therefore, the laboratory should conduct the necessary performance evaluation of the selected novel coronavirus antibody, carefully interpret the results of the novel coronavirus antibody, make the necessary further testing requirements, and reduce the missed diagnosis and misdiagnosis. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 2, 2020. . https://doi.org/10.1101/2020.04.27.20074849 doi: medRxiv preprint Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster Hypothesis for potential pathogenesis of SARS-CoV-2 Novel Corona Virus Pneumonia Diagnosis and Treatment Guideline (Trial Version 7) Detection of serum IgM and IgG for COVID-19 diagnosis. Science China Life sciences All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Viral Kinetics and Antibody Responses in Patients with COVID-19 Serological immunochromatographic approach in diagnosis with SARS-CoV-2 infected COVID-19 patients. The Journal of infection Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control Analytical performances of a chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG and antibody kinetics Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis Delivery in pregnant women infected with SARS-CoV-2: A fast review. International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics The project was supported partly by grants from National Natural Science Foundation of China (81572089), and partly by grants from Yuzhong District Scientific Research Project (20180129). Y.W. and P.L. conceived the project. Y.W., Z.L. and K.W. were responsible for the tissue sample collection. Y.W., Z.L. and T.L. performed the laboratory test. Y.W.conducted the statistical analysis. Y.W. and P.L. wrote the manuscript, and responsible for all data present in current research. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted May 2, 2020. . https://doi.org/10.1101/2020.04.27.20074849 doi: medRxiv preprint The authors declare no competing interests. The current investigation was conducted with the approval of the medical ethics committee of the General Hospital of Chongqing. Relevant study data will be made available on reasonable request from the corresponding author at liaopu2010@126.com All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted May 2, 2020. . https://doi.org/10.1101/2020.04.27.20074849 doi: medRxiv preprint All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted May 2, 2020. . https://doi.org/10.1101/2020.04.27.20074849 doi: medRxiv preprint