key: cord-0882166-h4holhit authors: Ling, R.; Yu, Y.; He, J.; Zhang, J.; Xu, S.; Sun, R.; Li, T.; Ji, H.; Wang, H. title: Seroprevalence and epidemiological characteristics of immunoglobulin M and G antibodies against SARS-CoV-2 in asymptomatic people in Wuhan, China date: 2020-06-19 journal: nan DOI: 10.1101/2020.06.16.20132423 sha: 374b91ddc6b514174525fb44e8c734d31b7b4610 doc_id: 882166 cord_uid: h4holhit Background The seroprevalence of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be a more reliable approach to detect true infected population, particularly in asymptomatic persons. Few studies focus on the diagnosis of COVID-19 patients using serological tests. To detect and assess asymptomatic infections of COVID-19 among people in Wuhan, the epicenter of the COVID-19 pandemic in China, and provide evidence for planning adequate public health measures, we collected and analyzed the clinical data in the Wuhan General Hospital mandatory for 16- to 64-year-old asymptomatic people. This retrospective study estimated the seroprevalence of IgM and IgG and compared the epidemiological characteristics of asymptomatic SARS-CoV-2-infected population. Methods Demographical and radiological data were collected from the Wuhan General Hospital between March 26 and April 28, 2020. Serological tests for IgM and IgG antibodies against SARS-CoV-2 were conducted with a colloidal gold method. Nucleic acid sequences of viruses were detected with RT-PCR. Statistical analyses were carried out using SPSS 20.0 software. Findings Between March 26 and April 28, 2020, 18,391 asymptomatic back-to-work participants were enrolled. Among them, 89 had positivity for IgM (0.48%, 95% confidence interval (CI): 0.38-0.58%); 620 cases had IgG positivity (3.37%, 95% CI: 3.11-3.64%), and 650 cases had either IgG positivity or IgM positivity (3.53%, 95% CI: 3.26-3.80%). After standardizing for the genders and ages in the population of Wuhan, the overall standardized seroprevalence of IgG was 3.33% (95% CI: 3.07-3.59%) and the standardized seroprevalence of IgG was 3.01% (95% CI: 2.69-3.33%) among males and 3.66% (95 % CI: 3.23-4.09%) among females. The standardized seroprevalence of IgG was higher in women than in men with a significant difference ({chi}2 = 2,060.3, p < 0.01). By a detection method adjustment, the seroprevalence of IgG was 1.57% (95% CI: 1.39-1.75%) in all medical records, of which males were 1.96% (95% CI: 1.64-2.28%), and females were 1.19% (95% CI: 0.99-1.39%). The assay-adjusted seroprevalence of IgG was higher in women than in men, and the difference was significant ({chi}2 = 5,871.0, p < 0.01). The differences were significant for the seroprevalence of IgG among people who went back to work in different categories of workplace ({chi}2 = 198.44, p < 0.01). The differences in seroprevalence for IgG positivity or IgM positivity among people who went back to work in different urban and rural areas was also significant ({chi}2 = 45.110, p < 0.01). Calculated as IgG and/or IgM antibody positivity, the number of new infections was reduced by 64.8% from March 26 to April 28, 2020. Based on the census population aged 16-64 years in Wuhan in 2017, we estimated that 172,340 (95% CI: 157,568-187,112) asymptomatic people aged 16-64 years were infected with SARS-CoV-2 in Wuhan between March 25 and April 28, 2020. This estimate was 3.4-times higher than the officially reported 50,333 infections on April 28. Interpretation The seropositivity rate in Wuhan indicated that RT-PCR-confirmed patients only represented a small part of the total number of cases. Seropositivity progressively decreased in the Wuhan population from March 26 to April 28, 2020, comparable to Japan and Denmark, but well below the level reported in New York, Iran, Italy, and Germany. The prevalence of asymptomatic infection was higher in women than in men among people who went back to work in Wuhan. The low seroprevalence suggests that most of the population remains susceptible to COVID-19. Funding The Emergency Management Project of the National Natural Science Foundation of China (81842035) and Advisory Research Project of the Chinese Academy of Engineering in 2019 (2019-XZ-70). The first case of Coronavirus Disease 2019 (COVID- 19) , which is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was reported on December 8, 2019, in Wuhan, Hubei Province. 1, 2 The Chinese public health, clinical, and scientific communities promptly responded in timely recognition of the new virus and shared the viral gene sequence to the world. 3, 4 The World Health Organization (WHO) termed the disease COVID-19 in January 2020. 2 The pandemic is the third coronavirus outbreak in the last 20 years, with severe acute respiratory syndrome caused by SARS-CoV and the Middle East respiratory syndrome by MERS-CoV. 5 As of May 17, 2020, COVID-19 infections occurred globally at more than 4,657,000 human cases and resulted in more than 312,000 human deaths according to the Johns Hopkins University. 6 The clinical symptoms of SARS-CoV-2 infection vary from mild to the onset of severe acute respiratory distress syndrome. Many patients can be asymptomatic, greatly increasing the uncertainty of diagnosis. 7 A timely and accurate diagnosis of COVID-19 is crucial for limiting further spread of the virus, particularly in people who are asymptomatic or mildly symptomatic, who can be responsible for community transmissions. 8 As Wuhan accepted the timely prevention strategies of lockdown and curbing population flow, it controlled the spread of the pandemic effectively. Nonetheless, there is still a risk of a second outbreak. Therefore, knowing the situation of SARS-CoV-2 infection in the general population is necessary. This knowledge has a meaning for the control and prevention of COVID-19 and epidemiological investigations, the determination of postdisease immunity and the use of future vaccine strategies. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) analysis for SARS-CoV-2 is the gold standard for COVID-19 diagnosis. [9] [10] It is time-consuming and requires special equipment with skilled laboratory technicians and has the weakness that easily leads to false negatives for SARS-CoV-2. [9] [10] [11] We know SARS-CoV-2-specific antibodies appear around 3-10 days after infection, with IgM appearing first and IgG following around 14 days after infection. 12 Even after the symptoms of infection disappear, IgG can still show seropositivity. 13 Therefore, SARS-CoV-2 serological assays are a good supplementary method for nucleic acid detection. 14 Additionally, serological assays allow studying the immune response(s) to SARS-CoV-2 in a dynamic qualitative and quantitative manner. In addition, serological antibody assays can determine the precise rate of infection in an affected area or a certain population, estimate the cumulative incidence and infection fatality rate, and provide parameters for epidemiological models to assess the possible impact of specific interventions, thus guiding public health decision-making. Furthermore, serological assays can be used to elucidate the geographic spread of the virus and identify subgroups of individuals who are more vulnerable to infection. Last, the degree of protection of antibodies against SARS-CoV-2 infection or reinfection can be determined by serological assays. 15,16 Therefore, testing for antibodies specific to SARS-CoV-2 protein could be an alternative method for rapid laboratory diagnosis with high sensitivity. 12 WHO recommends monitoring changes of seroprevalence over time, which is crucial at the beginning of an epidemic to anticipate and plan an adequate public health response. 9 At present, relatively few domestic and foreign reports exist on the diagnosis of patients with COVID-19 using serological tests. Thus, we conducted a retrospective study analyzing 18,391 medical 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. Hubei province including IgM and IgG tests for SARS-CoV-2 antibody, nucleic acid tests from March 26 to April 28, 2020 among people aged 16-64 years who went back to work. Current methods for measuring serum IgM and IgG mainly include enzyme-linked immunosorbent assays, chemiluminescence enzyme immunoassays, lateral flow immunoassays (LFIAs), and fluorescence immunoassays. LFIA specificity can reach about 99% with sensitivity from 80% to 86%. Combined IgG and IgM tests seem to be better choices for sensitivity than measuring either antibody type alone. 17 In our study, the LFIA method had a similar sensitivity and specificity for detecting SARS-CoV-2-specific antibodies. This study provides a reference for estimating the number of asymptomatic examples of COVID-19, the transmission patterns of the virus, and pandemic prevention strategies. This was a cross-sectional, retrospective study to investigate seroprevalence in asymptomatic COVID- A total of 17,608 people received nucleic acid tests, with one suspected as nucleic acid-positive, and positive for IgG antibodies and negative for IgM antibodies. Among the 783 people without nucleic acid tests, 10 (1· 28%) were positive by antibody tests and the rest were negative (98· 7%). A total of 18,391 people met the inclusion criteria with a median age of 40·0 years with 11,177 (60· 8%) males with median age 42·0 years and 7,214 (39·2%) females with median age 37·0 years. Clinical data were collected from March 26 to April 28, 2020, including serum IgG positivity and IgM positivity or negative results for SARS-CoV-2 antibodies, nucleic acid testing, clinical symptoms, previous medical history, and chest CT. SARS-CoV-2 antibody detection kits (colloidal gold method) were provided by INNOVITA (Tangshan, registration certificate for the medical devices of Peoples Republic of China: 20203400177). 18 All enrolled medical examiners collected 2 ml peripheral venous blood. After centrifugation, serum was taken for SARS-CoV-2-specific antibody IgG and IgM detection within 2 hours. Nasopharyngeal or oropharyngeal swabs used for SARS-CoV-2 nucleic acid testing were collected by trained and qualified medical staff for performing nucleic acid tests within 2 hours. The automatic nucleic acid extraction instrument was provided by the Hangzhou Allsheng Instruments Co. Ltd. (Allsheng). The ABI-7500 fluorescence PCR instrument was from Thermo Fisher Scientific Inc. All operations were carried out according to kit instructions. 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. percentile) was used for non-normal distribution data. P < 0.05 was considered statistically significant. To correct for the effects of gender and age structure, the seroprevalence of IgM positivity, IgG positivity, and IgM and/or IgG positivity were standardized according to the gender and age-specific population of Wuhan in 2017 (Attachment 2). 19 To correct for effects from accuracy of the serum antibody test, seroprevalence was corrected according to the sensitivity and specificity of the colloidal gold test in previous studies 13 following the Rogan Gladen methods. 20 This study was approved by the Ethics Committee of the Hubei Provincial Hospital of Integrated Chinese & Western Medicine. This was a retrospective and observational study and informed consent was obtained. Table 1 shows the unadjusted and adjusted seroprevalence for IgG and IgM against SARS-CoV-2 in asymptomatic people aged 16 to 64 by gender. The unadjusted seroprevalence of IgG in females was higher than in males and was significantly different (x 2 = 6·218, p < 0·05). The unadjusted seroprevalence of IgM in females was slightly higher than in males, but was not significantly different (x 2 = 0·207, p > 0·05). When IgG and IgM positivity were analyzed together, the unadjusted seroprevalence of IgG positivity and /or IgM positivity in females was higher than in males and was significantly different (x 2 = 6·865, p < 0·01). After standardization according to the numbers of people of different ages and genders in the population of Wuhan from the national census of 2017, the age-and gender-adjusted seroprevalence of IgG was 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 June 19, 2020. . https://doi.org/10.1101/2020.06.16.20132423 doi: medRxiv preprint higher in females than in males and the differences were significant (x 2 = 2,060·3, p < 0·01). After adjusting for ages and genders, and assay sensitivity and specificity, including CI, the overall adjusted seroprevalence of IgG was higher in females than in males. The difference between genders was significant (x 2 = 5,871·0, p < 0·01). The age-and gender-adjusted seroprevalence of IgG positivity and /or IgM positivity was higher in females than in males, and the difference was significant (x 2 = 979·26, p < 0·01). After adjusting for ages and genders, and assay type, the overall adjusted serum rate for IgG and /or IgM antibodies was 2·80 (95% CI: 2·56-3·04%) with 2·40 (95% CI: 2·11-2·68) for males and 3·23 (95% CI: 2·82-3·64) for females. Numbers were higher for females than males, and the difference was significant Table 2 . A significant difference was seen in seroprevalence of IgG among people from different units (x 2 = 198·44, p < 0·01), with no significant difference for IgM (x 2 =28·124, p>0·05), and a significant difference for IgG positivity and /or IgM positivity (x 2 = 200·21, p < 0·01). Among the tested individuals, the seroprevalence of IgG was highest for those who voluntarily came for examinations, followed by those reinforced by their work units, including real estate property companies, urban construction companies, telecommunications companies, banks, securities and insurance companies, law firms, and procuratorates. A significant difference was seen in seroprevalence of IgG by urban area (x 2 = 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 June 19, 2020. . https://doi.org/10.1101/2020.06.16.20132423 doi: medRxiv preprint 43·660, p < 0· 01), with no significant difference for IgM (x 2 = 14·783, p > 0·05), and a significant difference for IgG and/or IgM (x 2 = 45·110, p < 0·01). The Wuchang and Jianghan districts had higher seroprevalences of IgG than other urban areas. Detection of asymptomatic or subclinical novel human coronavirus SARS-CoV-2 infections is critical for understanding the overall prevalence and infection potential of COVID-19 21 According to a study in Denmark, 9,496 blood donors (17-69 years old) who gave blood during April 6-17, 2020 were tested for SARS-CoV-2 IgM and IgG antibodies using a commercial lateral flow test. The seroprevalence adjusted for assay sensitivity and specificity was 1·7% (CI: 0·9-2·3%), and differed by region. 22 A similar cross-sectional study conducted in Japan had an IgG seroprevalence of 3·3% (95% CI: 2·3-4·6%) using 1,000 samples from patients at outpatient settings. After adjusting for ages and genders, the IgG seroprevalence was 2·7% (95% CI: 1·8-3·9%), and the estimated number of infections was 396 to 858 times the number of confirmed cases at the time. 23 The seroprevalence estimated in our study was adjusted by age and gender, and then for assay sensitivity and specificity, which was comparable to the studies in Japan and Denmark, but lower than preliminary findings for Iran of 22%, New York City of around 14%, Italy of 10%, and Germany of 14%. [24] [25] [26] [27] All these studies indicated that the number of asymptomatic infections was much higher than the number of confirmed cases reported based on PCR results. Our study found that the number of new infections was reduced by 64· 8% to a relatively lower level of 1·46 (95% CI: 0·93-1·98%) of the IgG seroprevalence among the urban population aged 16-64 years on (Attachment 2). Of note, the officially reported number included people under age 16 and over age 64, yet was lower than the calculated number in this study, implicating underestimation due to omitted detection of asymptomatic infections. Data from a detailed surveillance study from the Chinese Center for Disease Control and Prevention indicates that 80% of confirmed cases were mild, and a number of patients can remain asymptomatic with very low symptomatology. 28 Thus, epidemiological studies of asymptomatic infections should receive sufficient attention. Some studies find an average level of IgG antibodies that is higher in females than in males. 29 In our 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 June 19, 2020. . https://doi.org/10.1101/2020.06.16.20132423 doi: medRxiv preprint study, the IgG seroprevalence was higher in females than in males, indicating that women were more likely to have asymptomatic infections. People living or working in the Wuchang and Jianghan districts had a higher IgG seroprevalence of 4·60 (95% CI:3·21-5·99) and 4·18 (95% CI:3·80-4·56) respectively than people from other districts of Wuhan in our study. Based on the cumulative number of reported cases of COVID-19 and the number of residents in each district in Wuhan on March 25 and April 28, we estimated an average incidence rate per 100,000 people of 2·98 (95% CI: 2·97-2·99) during this period. Incidence rates were higher in the Jianghan and Wuchang districts, at 8·09 (95% CI: 8·02-8·15) and 7·25 (95% CI: 7·20-7·29) per 100,000 people, respectively, which were the same as our regional distribution analyses (Attachment 2). Our study found significant differences in IgG seroprevalence among people going back to work by types of jobs, with the highest seroprevalence in volunteer medical examinees who came for tests without a mandate from their job supervisors. These self-motivated individuals had close contacts with COVID-19 patients, and therefore, had a higher risk of infection. Other individuals with high IgG seroprevalence were easy to identify due to the nature of their jobs. They undertook substantial voluntary work in response to government calls during the COVID-19 pandemic, to assist in, for instance, administering in the local residential communities, transporting COVID-19 patients, and screening the general population for epidemic incidence. Thus, they faced a greater chance of contacting infected people during their work, and were more likely to be infected, even with personal protection. This study had a few limitations. First, this study had selection bias since the analyzed medical records were based on examinees directed by their work units. Most of the examinees came from governmentowned institutions and agencies instead of private businesses. Therefore, the sample was incompletely randomized and insufficiently representative, compromising the assessment accuracy of the prevalence of asymptomatic infections in Wuhan. Second, as the examinees were only from the back-to-work population, people under age 16 and over age 64 were not covered in analyses. Therefore, we could not estimate COVID-19 in these two groups. Our study provides information to herd immunity investigation to help design targeted strategies for prevention. Research on serological antibodies has great value for COVID-19 prevention. 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. previous studies following the Rogan Gladen methods, to correct for effects from accuracy of the serum antibody test. According to the Rogan Gladen methods, P=(t + β -1)/(α + β -1), in which P is adjusted seroprevalence, α is sensitivity of the antibody test (colloidal gold method ), β is specificity of the antibody test by colloidal gold method, t is age-and gender-adjusted seroprevalence or positive test frequency. 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. 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. N is the total number of individuals, n1 is the number of IgG-positive individuals, n2 is the number of IgM-positive individuals, n3 is the number of IgGpositive or IgM-positive. Real estate includes real estate agencies, residential property management firms, and urban construction corporations. 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 June 19, 2020. . https://doi.org/10.1101/2020.06.16.20132423 doi: medRxiv preprint Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study An Invited Commentary on "World Health Organization declares global emergency: A review of the 2019 novel Coronavirus (COVID-19)": Emergency or new reality? A Novel Coronavirus from Patients with Pneumonia in China A new coronavirus associated with human respiratory disease in China The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak COVID-19 Case Tracker The Incubation Period of Coronavirus Disease From Publicly Reported Confirmed Cases: Estimation and Application Estimating the asymptomatic proportion of coronavirus disease 2019 (COVID-19) cases on board the Diamond Princess cruise ship Population-based age-stratified seroepidemiological investigation protocol for COVID-19 virus infection Molecular and serological investigation of 2019-nCoV infected patients: implication of multiple shedding routes. Emerging microbes & infections 2020 Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019. 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