Laboratory Medicine Network
The following article is from the Chinese Journal of Laboratory Medicine, by Wanzhou Xu, et al.
Chinese Journal of Laboratory Medicine
Chinese Journal of Laboratory Medicine
This public number mainly publishes information related to laboratory medicine, serving the majority of senior laboratory technicians, medical laboratory researchers and clinical physicians. The Chinese Journal of Laboratory Medicine was founded in September 1978, and has been awarded the title of "100 Chinese Outstanding Academic Journals" for many times, and has been listed in the "Chinese Journal Square", and has been awarded the Excellent Journal Award by the Chinese Medical Association.
Authors：Xu Wanzhou1 Li Juan1 He Xiaoyun1 Zhang Caiqing1 Mei Siqing1 Li Congrong1 Li Yan1 Cheng Shaohua2 Zhang Pingan1
Affiliations：1-Department of Laboratory Science, Wuhan University People's Hospital, Wuhan, China 2-Quality and Safety Management Office, Wuhan University People's Hospital, Wuhan, China
Corresponding author: Pingan Zhang Shaohua Cheng
◆◆ Abstract◆◆
Objective
To explore the diagnostic value of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibody tests for novel coronavirus (2019-nCoV) in novel coronavirus infection.
Methods
In this study, 284 outpatients and inpatients attending the People's Hospital of Wuhan University from January 20, 2020 to February 17, 2020 were collected using a retrospective study, of which 205 were patients with novel coronavirus pneumonia, including 186 confirmed patients with positive 2019-nCoV nucleic acid test and 186 patients with negative 2019-nCoV nucleic acid test but clinical symptoms and CT The test results were consistent with the Novel Coronavirus Pneumonia Prevention and Control Program (5th edition) in 19 patients as the case group. Seventy-nine patients with other diseases excluding novel coronavirus pneumonia were used as the control group. Serum 2019-nCoV IgM and IgG antibodies were detected by fully automated chemiluminescent immunoassay technique in all study subjects. The differences in the results of 2019-nCoV IgM and IgG antibody tests and nucleic acid tests were statistically analyzed using the χ2 test.
Results.
The clinical sensitivity of serum 2019-nCoV IgM and 2019-nCoV IgG was 70.24% (144/205) and 96.10% (197/205), respectively, and the clinical specificity was 96.20% (76/79) and 92.41% (73/79), respectively. the positive predictive value of 2019-nCoV antibody test was 95.63% (197/206) and 91.03% (71/78) for negative predictive value, and 100% (186/186) for positive predictive value and 80.61% (79/98) for negative predictive value for 2019-nCoV nucleic acid test. the overall compliance rate for 2019-nCoV antibody test and 2019-nCoV nucleic acid to diagnose 2019-nCoV infection was 88.03% (250/284).
Conclusion
The combined serum 2019-nCoV IgM and IgG test can be used as a valid screening and diagnostic indicator for novel coronavirus infection and is an effective complement to false-negative nucleic acid testing for novel coronavirus pneumonia.
Annex 4 of the Novel Coronavirus Pneumonia Prevention and Control Program (Fifth Edition), published by the National Health and Wellness Commission on February 21, 2020, entitled "Technical Guidelines for Laboratory Testing of Novel Coronavirus Pneumonia" [1], states that the application of novel coronavirus (2019 Novel Coronavirus, 2019-nCoV) antibody testing can be used as a novel coronavirus pneumonia (COVID- 19) as an aid in the diagnosis of the disease.
After the virus infects the organism, the immune system performs immune defense against the virus and produces specific antibodies. IgG antibodies are the primary antibodies produced in response to a renewed immune response and are indicative of recovery or the presence of a previous infection.
Therefore, the combination of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies can not only provide early diagnosis of infectious diseases, but also help to assess the stage of infection in the body. In this study, serum 2019-nCoV IgM and IgG antibodies were detected by fully automated chemiluminescent immunoassay technique and their laboratory diagnostic value in COVID-19 was evaluated.
◆◆Subjects and methods◆◆
1Subjects
284 outpatients and inpatients attending the People's Hospital of Wuhan University from January 20, 2020 to February 17, 2020 were collected, including 205 cases in the case group, 88 males and 117 females, aged 23 to 87 years, all of whom were COVID-19 patients, including 186 confirmed patients with positive 2019-nCoV nucleic acid test and 2019-nCoV nucleic acid test negative but clinical symptoms and CT test results were consistent with the Novel Coronavirus Pneumonia Prevention and Control Program (5th edition) in 19 patients. The control group consisted of 79 patients, 38 males and 41 females, aged 24 to 101 years, who were patients with other diseases excluding COVID-19. The study complied with the International Coordinating Council clinical trial specifications and the Declaration of Helsinki, and was reviewed and approved by the hospital ethics review committee (ethics number: WDRY2020-K30), and approved to exempt patients from informed consent.
2 Methods
1. Reagents and instruments: 2019-nCoV IgM and IgG antibody chemiluminescence detection kits from Shenzhen Yafei Long Biotechnology Co., Ltd. and magnetic particle-coated antigens including 2019-nCoV stinging protein S and nucleocapsid protein N antigens. iFlash3000 automatic chemiluminescence immunoassay analyzer from Shenzhen Yafei Long Biotechnology Co. The magnetic bead extraction kit was obtained from Ningbo Hilshi Gene Technology, and the Labassist32 automated nucleic acid extractor was obtained from Taiwan Round Point Nanotechnology Development Co. Novel coronavirus (2019-nCoV) ORF1ab/N gene dual nucleic acid detection kit (fluorescence PCR method) was from Shanghai JENO, and QuantstudioDx and 7500 fluorescence PCR instruments were purchased from Thermo Fisher, USA.
2. Specimen collection: 5 ml of fasting venous blood was collected from all study subjects, placed in a yellow-tipped vacuum blood collection tube containing separation gel, left to clot, centrifuged at 2500×g for 5 min, and the serum was taken as a backup. Nasopharyngeal swab specimens were collected from the study subjects for 2019-nCoV nucleic acid detection.
3. Serological detection: serum 2019-nCoV IgM and IgG antibodies were detected by fully automated chemiluminescence immunoassay technique, and the detection steps were referred to the kit instructions, and the test results were expressed as relative light unit (RLU). The amount of 2019-nCoV IgM or IgG antibody in the sample and the RLU are positively correlated, and the assay automatically calculates the 2019-nCoV IgM or IgG antibody concentration (AU/ml) based on the RLU and the built-in calibration curve, and the test result of 10.0 AU/ml is considered reactive (positive).
4. 2019-nCoV nucleic acid detection: fluorescence RT-PCR was used to detect open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) in the 2019-nCoV genome. The interpretation of the value is based on the recommendation of each manufacturer's instructions, and the clinic should be notified to resample and recheck the suspicious results. In the laboratory test results to confirm a positive diagnosis, it is necessary to meet the same specimen of 2019-nCoV ORF1ab and N gene at least 1 target-specific RT-PCR test results are positive.
5. statistical analysis: SPSS 19.0 software was used for data processing. The survey data were counted, percentages and line list statistics were selected, and the differences in sensitivity, specificity, positive predictive value and negative predictive value of 2019-nCoV nucleic acid and antibody tests for the diagnosis of 2019-nCoV infection were tested by χ2 test. The difference was considered statistically significant at P<0.05.
◆◆Results◆◆
I. Clinical specificity of 2019-nCoV IgM and IgG antibody assay
Among the 79 patients in the control group, 76 cases of 2019-nCoV IgM antibody were negative and 3 cases were false positive, with a clinical specificity of 96.20% (76/79), 73 cases of 2019-nCoV IgG antibody were negative and 6 cases were false positive, with a clinical specificity of 92.41% (73/79), among which 1 of the 3 cases of false positive 2019-nCoV IgM antibody Among the 3 false positive 2019-nCoV IgM antibody cases, 1 case had a high 2019-nCoV IgM level of 68.28 AU/ml, which was a 93-year-old patient with hemorrhagic shock, and the remaining 2 cases showed weak positivity with results of 14.46 AU/ml and 20.27 AU/ml, respectively, both being tumor patients. The rest were within the range of 10.03 AU/ml to 14.49 AU/ml, which were weakly positive reactions. 2019-nCoV IgM and IgG antibody test false positives may be caused by autoantibodies, heterophilic antibodies and other factors. Therefore, the 2019-nCoV IgM and IgG antibody test reagent has high clinical specificity and can meet the screening and diagnostic requirements of COVID-19.
II. Clinical sensitivity of 2019-nCoV IgM and IgG antibody assay
Among 205 COVID-19 patients, 144 were positive for 2019-nCoV IgM antibody and 61 were negative, 197 were positive for 2019-nCoV IgG antibody and 8 were negative. the clinical sensitivity of 2019-nCoV IgM and IgG for 2019-nCoV infection was 70.24% (144/205) and 96.10% ( 197/205). The results indicate that the combined 2019-nCoV IgM and IgG antibody test has a very high detection rate in COVID-19 patients and is a good complement to nucleic acid detection for missed tests.
III. Comparison of 2019-nCoV antibody and 2019-nCoV nucleic acid detection
The results of 2019-nCoV IgM and IgG antibody tests and nucleic acid tests in 284 study subjects were compared, as shown in Table 1. the positive predictive value of 2019-nCoV antibody test was 95.63% (197/206) and the negative predictive value was 91.03% (71/78), and the positive predictive value of 2019-nCoV nucleic acid test was 100% ( 186/186) and a negative predictive value of 80.61% (79/98). the overall compliance rate for the diagnosis of 2019-nCoV infection by 2019-nCoV antibody and 2019-nCoV nucleic acid was 88.03% (250/284). The difference between the two indices for the diagnosis of 2019-nCoV infection was statistically significant by χ2 test (χ2=151.99, P<0.05). Serological testing of 19 clinically confirmed patients with clinical symptoms of COVID-19 and CT imaging features but negative nucleic acid tests revealed positive reactions for 2019-nCoV IgM antibodies in 16 cases (84.21%) and positive reactions for 2019-nCoV IgG antibodies in 18 cases (94.74%).
◆◆ Discussion◆◆
The pneumonia epidemic caused by 2019-nCoV spreads rapidly and poses a serious threat to people's life safety and health[2-3] .2019-nCoV belongs to the genus β coronavirus with a linear genome of single-stranded positive-stranded RNA and is the seventh coronavirus known to infect humans[4] . Currently, 2019-nCoV nucleic acid testing is the routine test and the basis for confirming the diagnosis of COVID-19. The latest "Pneumonia diagnosis and treatment protocol for novel coronavirus infection (trial version 6)" published by the National Health and Wellness Commission on February 18, 2020, suggests that a positive nucleic acid test or viral gene sequencing with a high degree of homology to a known novel coronavirus is required for a confirmed case [5].
However, not all 2019-nCoV-infected patients can be clinically tested for 2019-nCoV nucleic acid by routine specimens, and false-negative 2019-nCoV nucleic acid tests have been observed in several clinical institutions in multiple locations, resulting in nucleic acid test results that are inconsistent with clinical symptoms and imaging examinations. This involves various factors such as sample collection and storage, site of viral infection, RNA extraction method, and quality of nucleic acid detection kits [6-7], which brings great impact on the diagnosis of 2019-nCoV infection and epidemic prevention and control.
In this context, the national pneumonia diagnosis and treatment protocol for novel coronavirus infections (trial version 5) [8] was published, stating that patients who are nucleic acid negative but have specific clinical symptoms should be identified as "clinically confirmed cases", thus greatly reducing the deterioration of suspected cases and the potential risk of infection to healthy people.
Although the Novel Coronavirus Pneumonia Prevention and Control Program (5th Edition) is an interim program, the addition of clinical diagnosis criteria during the progressive recognition of the disease has played an undeniable role in the control of the COVID-19 outbreak at this particular time. In particular, Annex 4 of the Novel Coronavirus Pneumonia Prevention and Control Program (Fifth Edition), Technical Guidelines for Laboratory Testing of Novel Coronavirus Pneumonia [1], emphasizes the necessity of positive pathogenic testing for the confirmation of COVID-19 diagnosis, and also points out that negative nucleic acid test results cannot exclude novel coronavirus infection, and proposes the use of serum samples for antibody testing, which should be considered to be a contribution to the Novel Coronavirus Pneumonia Treatment Protocol for Novel Coronavirus Infection (Trial Version 6)" for the exclusion of suspected cases.
Since late February 2020, some domestic reagent manufacturers have developed immunoassay kits for 2019-nCoV IgM and IgG antibodies one after another. Antibody detection is a new test for 2019-nCoV, and it is very necessary to fully validate the clinical specificity and clinical sensitivity of COVID-19 serological laboratory diagnosis, especially to investigate whether antigen-antibody serological testing can play a significant complementary role to the problem of nucleic acid miss detection. The study showed that the 2019-nCoV IgM and IgG immunoassay reagents have high clinical specificity, reaching 96.20% and 92.41%, respectively, and can fully meet the needs of clinical testing. 2019-nCoV IgM assay has a clinical sensitivity of 70.24% in COVID-19 cases, while its IgG assay has a clinical sensitivity of 96.10%, which can be effectively used as screening and diagnosis of novel coronaviruses.
In this study, we found that all 2019-nCoV Ig
