Surveillance of SARS-CoV-2 Antibody of Patients in Local Affected Area During the Lockdown

Background: Serosurvalence is crucial in estimating the range of SARS-CoV-2 infections, predicting the possibility of another wave, and decide on a vaccination strategy. To understand the herd immunity after the COVID-19 pandemic, the seroprevalence was measured in 3062 individuals with or without COVID-19. Methods: The levels of SARS-CoV-2 antibody IgM and IgG were measured by the immuno-colloidal gold method. Results: The mean seroprevalence for IgM and IgG in all participants was 2.81% and 7.51%, respectively. The positive rate of IgG was signicantly higher in women than in men (P<0.05). The highest positive rate of IgM was observed in 41-60 years of age (3.49%), while the highest seroprevalence for IgG was observed in persons >60 years of age (8.61%). The positive rates of IgM and IgG in the convalescent patients were 31.82% and 77.27%, respectively, which was signicantly higher than individuals with suspected syndromes or individuals without any clinical signs (P<0.01). Seroprevalence for IgG in medical staff was markedly higher than those in residents. The seroprevalence in patients with various comorbidity was no signicant difference (P>0.05). Conclusions: The low positive rate of the SARS-CoV-2 IgM and NA test indicated that the SARS-CoV-2 outbreak is subsiding after three months, and the possibility of reintroduction of the virus from an unidentied natural reservoir is low. Seroprevalence provides the information for humoral immunity and vaccine in the future.

Serosurvalence is crucial in estimating the range of SARS-CoV-2 infections, predicting the possibility of another wave, and decide on a vaccination strategy.
Neutralizing antibodies (NAbs) are critical components in the protective immune responses to viral infections because they can bind to viral particles and block them from entering the host cells (7,8). NAbs are essential for protecting populations from re-infection. Information on the NAbs could be used to understand the epidemiology of SARS-CoV-2 infection and help determine the level of humoral immunity in patients. To COVID-19, different populations in different regions may have different humoral immunity.
Wuhan was the epicenter of the COVID-19 of China, with the highest infectious rates; residents who lived in this city should have a high-risk for virus exposure. However, few data report residents' infection with certainty. As a tertiary university medical center in metropolitan Wuhan, Zhongnan hospital has a 3300bed capacity, and it serves about 100,000 people. Zhongnan hospital was designated a hospital responsible for COVID-19 patients' treatment during the pandemic. Many severe and critically severe patients were transferred there for intense therapy. Then, we selected this hospital to study the prevalence of COVID-19 infection. Given the relatively extraordinary exposure history of the individuals, including patients and hospital staff of this hospital, their seroprevalence may provide valuable information about the population infectious and their immune status. Seroprevalence of residents is vital for understanding the infectious population scale and their immune status and preventing disease spread and reemergence.
Methods: Sample collection.
The study received ethics approval from the Ethics Committee of Zhongnan Hospital, Wuhan University. The blood samples were collected from a total of 3062 outpatients, including 2597 ordinary patients for COVID-19 screening, 355 individuals with suspected clinical symptoms, and 110 con rmed COVID-19 patients whose diagnosis was de ned based on the New Coronavirus Pneumonia Prevention and Control Program (7th edition) published by the National Health Commission of China. Blood samples were obtained in containers without anticoagulant and kept at room temperature for 0.5 h to ensure serum separation. Serum samples were collected after being centrifuged at 4,000 rpm for 5 min. Whether the participants had been exposed or the time of their exposure cannot be known with certainty. Therefore, a single blood sample was taken for antibody testing. Blood samples were collected after March 21, 2020, approximately two months after the outbreaks were recognized.

Antibody and nucleic acid of SARS-CoV-2 Detection
Serum IgM and IgG of SARS-CoV-2 was measured by immuno-colloidal gold technology (INNOVITA Biotechnology Company, Tangshan, China). 10uL serum samples diluted with two drops of sample diluent and then added in sampling well. Read the result within 15min. Both the control line and the test line appear simultaneously, indicating a positive outcome. Only show the control line, the result is negative, and if the control line does not appear, the result is invalid. At the same time, the nucleic acid (NA) of SARS-CoV-2 was measured by reverse transcription-polymerase chain reaction (RT-PCR) (DAAN GENE Company, Guangzhou, China). Also, all participants underwent a CT scan to con rm whether there were pathogenic lesions in the lung.

Statistical analyses.
Data were expressed as numbers and proportions. Differences in frequencies or proportions were tested using a χ2 test. SPSS 20.0 was used for statistical analyses (SPSS Incorporated, Chicago, IL, USA). P < 0.05 were statistically signi cant.

Results:
In total, 3062 individuals were enrolled in this study, of whom 1652 were males, and 1410 were females. Of 3062 samples, seroprevalence for IgM and IgG were 86(2.81%) and 230 (7.51%), respectively. In persons with seropositive, 97 persons had positive CT scan results (3.16%); only four persons (0.13%) had NA positive, including two convalescent COVID-19 patients recovered in the observation period and two asymptomatic infection cases. The rate of speci c antibody IgG was signi cantly higher in women than in men (P < 0.05).
The variation of IgM and IgG in COVID-19 patients.
The percentage of patients who were IgM positive reduced after March 22, keeping at a low level after that. Seroprevalence for IgG peaked at 25.93% on March 23 due to many COVID-19 patients tested the NAbs on those days. As time passed, the positive rates of IgG remained at a low level; the range was from 2.82-14.08% (Fig. 1A). Most patients were convalescent, and the positive rate of IgG was greater than that of IgM (Fig. 1A). As for subgroups, the positive rate of IgM had a higher level in the COVID-19 convalescent group (mean ± SE: 33.41 ± 0.34) than SSC (mean ± SE: 4.41 ± 0.09) and SAC groups (mean ± SE: 1.28 ± 0.09). During all study periods, the COVID-19 convalescent group showed a higher IgG positive rate (mean ± SE: 81.19 ± 0.26) than SSC (mean ± SE: 7.35 ± 0.05) and SAC groups (mean ± SE: 4.62 ± 0.09) (Fig. 1B).

Discussion:
Due to its extreme high contagious, COVID-19 elicited a signi cant threat to global public health. It is of utmost importance to know whether the SARS-CoV-2 outbreak is subsiding after tremendous efforts on interrupting the human-to-human transmission to reduce secondary infections among close contacts and to prevent transmission ampli cation events. Currently, the rst epidemic of COVID-19 is under control in Wuhan. Much work followed should be on preventing asymptomatic transmission. Some asymptomatic individuals might have existed; a new reservoir of the virus may exist that signals the need for continued surveillance. To investigate the humoral responses to the virus in the context of epidemiologic settings in the epicenter of COVID-19, we randomly selected individuals with or without suspected syndromes and convalescent COVID-19 patients from different consulting departments. Our results revealed a low positive rate of NA tests in the studied cohort. Seroprevalence for IgM and IgG in 355 individuals with suspected syndromes were signi cantly higher than those of 1295 asymptomatic participants (IgM and IgG were 3.38% vs. 1.70% and 7.89% vs. 5.17%, respectively), indicating that viral infection in some cases only has mild or even no clinical manifestations. IgM is considered a parameter of the early phase of infection. IgM against SARS-CoV can be detected as early as in the rst week (9). In our study, seroprevalence for IgM in ordinary patients for COVID-19 screening was more sensitive than the result of NA tests that was almost negative in our participants; the reason may be due to short phases of the virus shedding, or insu cient sample quality decrease the chances of detecting nucleic acids (10). IgM detection assays are incredibly valuable because it helps to nd patients in the acute phase and to elucidate the range of subclinical infectious individual. In our study, the positive rate of IgM continues to decrease over time, consistent with a gradual decline in newly diagnostic cases. Combing to seroprevalence for IgM and positive rate of NA, the SARS-CoV-2 outbreak is subsiding, and the possibility of reintroduction of the virus from an unidenti ed natural reservoir will low.
The SARS-CoV-2 is transmitted by direct contact, droplets, feces, aerosols, or contaminated environmental surfaces. Therefore, the higher risk faced by a resident in the epicenter may be related in part to the temporal excretion pattern of the virus from COVID-19 patients. As we write this article, there is few data elucidate the infectious scale. Seroprevalence for IgG remained at the range was less than 10% in Wuhan residents, which is much lower than we expected. The nding of such low seroprevalence may be attributed to strictly precautionary measures, including home quarantine, temperature measurement, and must wear a surgical mask before entering the public place, washing hands frequently, etc. The highest seroprevalence for IgG was observed in COVID-19 convalescents. However, the positive rate of IgG was only 77.27% in convalescent patients. Eleven patients had IgG negative but IgM positive, suggesting they were newly infectious patients. Our data consistent with the previous reports, the presence of antibodies was < 40% among patients within 1-week since onset and rapidly increased to 94.3% (IgM) and 79.8% (IgG) since day-15 after onset (11).
In sixteen medical staffs who had suspected syndromes, the serological tests for IgM were entirely negative, the positive rate of IgG was 25%, and the positive rate of NA was 6.25%, indicating that latent infection had existed in these medical staffs. Seroprevalence for IgG in medical staff was signi cantly higher than those in residents. Before the outbreak was recognized, COVID-19 can be incredibly di cult to recognize due to the nonspeci c nature of clinical manifestations, which may be the leading cause of high seropositive in medical staff. After the outbreak announced, all doctors entering isolation areas were required to follow the SOP about attire and to equip themselves with a double layer of personal protective equipment (PPE), including an N95 mask, covered with a full-face mask, goggles, a long-sleeve gown, a paper hat, and shoe covers. Our results revealed that personal protection was effective against viral infection, even in high-risk viral exposure.
NAbs are vital components in the protective immune responses to viral infections (7,12). When evaluating the impact of comorbidity on immune responses, higher seroprevalence was observed in patients with tumors, cardiovascular and cerebrovascular diseases, hypertension, diabetes, nerve system disease, and digestive system diseases, but there was no signi cant difference among various groups. The seropositivity was higher in advanced age participants, the low prevalence in the younger populations.
Higher seroprevalence of IgG was in the female than in the male, which may be because a man had low adaptive immune responses than a woman (13). A signi cant nding of our study is that none of the AIDS patients were infected. Either IgM or IgG was negative. Since there were only 13 patients, we could not rule out a sampling error. To COVID-19, information on immunity and pathogenesis is insu cient to provide a comprehensive basis for a speci c drug or vaccine design. The observational data of NAbs may provide leads in controlling a possible reemergence of the disease.
Based on the transmission risk of known or unknown sources, infectious sources could not be ascertained. Patients without suspected syndromes were signi cantly more likely to be seronegative than those with the suspected syndrome. Moreover, participants who are come from the fever clinic had a signi cantly higher positive rate of IgM and IgG than other departments, demonstrating that the establishment of a fever clinic for suspected cases screening can improve the detection rate, achieve the goal of rst isolation. The nding in asymptomatic seropositive persons indicates that the test will be useful in more extensive retrospective surveillance studies, which are needed to de ne the epidemiology and spectrum of disease fully (14).

Conclusion:
In the later period of the epidemic, the potential reintroduction of the virus from an unidenti ed natural reservoir remains a concern. Our results suggested that sero-surveillance is particularly valuable to trace hotspots of persons carrying antibodies to SARS-CoV and to track the origins of the disease. The low rate of SARS-CoV-2 antibodies in Wuhan residents indicated that the chain of human transmission could be successfully interrupted by public health measures. TYT and XYJ designed and drafted the manuscript. YRL designed and revised the nal manuscript. YRL ,TYT, and BKZ collected and summarized the clinical laboratory and clinical data. XW, YG, and LZ performed specimen collection antibody detection experiments. GMY is responsible for summarizing all data related to the virus. JYS ,YF , TYT and YMY processed statistical data. YRL had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. YRL and XYJ contributed equally to this article and shared the corresponding authorship. All authors have read and approved the manuscript and ensure that this is the case.