Dynamic changes of IgM and IgG antibodies in asymptomatic patients as an effective way to detect SARS‐CoV‐2 infection

Abstract Background COVID‐19 has become a global pandemic, and close contacts and asymptomatic patients are worthy of attention. Methods A total of 1844 people in close contacts with 76 COVID‐19 patients were investigated, and nasopharyngeal swabs and venous blood were collected for centralized medical quarantine observation. Real‐time fluorescence was used to detect SARS‐CoV‐2 nucleic acid in nasopharyngeal swabs of all close contacts, and the colloidal gold method was used to detect serum‐specific antibodies. Levels of IgM‐ and IgG‐specific antibodies were detected quantitatively through chemiluminescence from the first nucleic acid turned negative date (0 week) and on weekly intervals of ≤1 week, 1–2 weeks, 2–3 weeks, 3–4 weeks, 4–5 weeks, 5–6 weeks, and 6–7 weeks. Results The total positive rate of the colloidal gold method (88.5%, 23/26) was significantly higher (χ2 = 59.182, p < 0.001) than that of the healthy control group (2.0%, 1/50). There was significant difference in IgG concentration at different time points (0–7 weeks) after negative nucleic acid conversion (χ2 = 14.034, p = 0.029). Serum IgG levels were significantly higher at weekly time points of 4–5 weeks (Z = −2.399, p = 0.016), 5–6 weeks (Z = −2.049, p = 0.040), and 6–7 weeks (Z = −2.197, p = 0.028) compared with 1–2 weeks after negative nucleic acid conversion. However, there was no significant difference (χ2 = 4.936, p = 0.552) in IgM concentration between time points tested (0–7 weeks) after negative nucleic acid conversion. The positive rates of IgM and IgG in asymptomatic patients (χ2 = 84.660, p < 0.001) were significantly higher than those in the healthy control group (χ2 = 9.201, p = 0.002) within 7 weeks of negative nucleic acid conversion. Conclusions The IgG concentration in asymptomatic cases remained at a high level after nucleic acid turned negative. Nucleic acid detection combined with IgM and IgG antibody detection is an effective way to screen asymptomatic infections.


| INTRODUC TI ON
In December 2019, an unexplained viral pneumonia was first reported in Wuhan, Hubei Province, China. 1,2 Subsequently, the disease was discovered in many countries and swept the globe. The novel coro- In most cases, COVID-19 patients were classified as asymptomatic, mild, moderate, and severe critical disease based on the clinical severity. 4 Among these, the highly contagious of asymptomatic patients with a long latent period and strong infectivity made control and prevention efforts of COVID-19 transmission extremely difficult. 5 Laboratory testing plays a crucial role in early diagnosis, severe evaluation, and prognostic treatment of the disease. Currently, the nucleic acid testing depending on the real-time PCR (RT-PCR) remains the gold standard for the diagnosis of SARS-CoV-2-infected patients. However, due to some limitations in nucleic acid testing, such as long detection cycle, expensive reagents and high requirements for sampling, and technicians and laboratory conditions, many COVID-19 cases are not diagnosed accurately and timely. 6,7 Up to now, 369 cases of asymptomatic infection in China were still under medical observation, including 352 cases imported from abroad. 8 Therefore, there is an urgent need to explore a rapid, simple, and feasible method for the diagnosis and screening of COVID-19 patients, especially the asymptomatic cases.
As generally known, serum immunoglobulin M (IgM) antibodies appear in the early stages of viral infection, followed by the production of serum immunoglobulin G (IgG) antibodies, which are essential for long-term immunity and immune memory. Furthermore, several studies have been reported that IgM and IgG antibodies can be detected in the first 1.5-8 days after the onset of symptoms, and may exist dynamically for a significant period of time. 9,10 Detecting serum IgM and IgG antibodies may provide a valuable detection method for the diagnosis and treatment of COVID-19, especially, when screening for asymptomatic infection. The aim of this study was to provide evidence for screening infection in asymptomatic patients with COVID-19 through the dynamic monitoring of IgM and IgG concentration levels.

| Patients
To identify asymptomatic individuals, the Disease Control and Prevention (CDC) of Loudi in China screened a total of 1844 close contacts (mean age 38.2 ± 19.9 years old), including 1003 men (mean age 38.7 ± 19.9 years old) and 841 women (mean age 37.7 ± 20.0 years), with 76 confirmed patients with COVID-19 from January to May 2020. Nasopharyngeal swabs were collected for SARS-CoV-2 nucleic acid detection from all close contacts. Participants then submitted to a 14-day medical quarantine observation period. Asymptomatic patients whose two consecutive nucleic acid tests were negative after

| Detection of SARS-CoV-2 nucleic acid in close contacts
Nasopharyngeal swabs were collected from 1844 close contacts and analyzed using a SARS-CoV-2 nucleic acid detection kit, following the manufacturer's instructions.

| Qualitative detection of serum SARS-CoV-2 antibodies in asymptomatic patients
Blood was drawn from asymptomatic patients and centrifuged for 10 min to obtain serum. We qualitatively detected SARS-CoV-2 antibody using the colloidal gold method.

| Quantitative and dynamic detection of serum SARS-CoV-2 IgM-and IgG-specific antibodies in asymptomatic patients
Pharyngeal swabs were collected for SARS-CoV-2 nucleic acid tests until tests were negative for three consecutive times. Blood was collected from the first nucleic acid negative date (0 week) within weekly periods: ≤1 week, 1-2 weeks, 2-3 weeks, 3-4 weeks, 4-5 weeks, 5-6 weeks, and 6-7 weeks. Blood was centrifuged at 1650 g for 10 min. Sera were collected to detect IgM and IgG levels quantitatively through chemiluminescence.

| Statistical analysis
Statistical analyses were performed using SPSS (v. 19.0) software with α of 5%. The measurement data are presented as mean ± standard deviation or median (interquartile range [IQR] P25, P75).
Comparisons between two groups were performed using a t test and Wilcoxon's rank sum test. Enumeration data are expressed as percentages using the χ 2 test.

| Epidemiology of close contacts
Among the 1844 close contact cases, the nucleic acid swabs for 33 individuals with no clinical symptoms were positive; these individuals were treated as asymptomatic infected patients and were placed under medical observation. One week later, 7 of them developed fever, cough, and other symptoms ( Figure 1  which ranged from 1-31 days, and the median communicable period (MCP) was 7.5 days.

| Qualitative detection of antibodies to identify asymptomatic infections
Blood was drawn from 26 asymptomatic infected cases for nucleic acid tests with positive results. Twenty-three of the 26 asymptomatic cases were positive for COVID-19-specific antibodies by the colloidal gold method, and only 3 cases were negative. The total positive rate of the colloidal gold method was 88.5% (23/26), which was significantly higher than that of the healthy control group (2.0%) (χ 2 = 59.182, p < 0.001). The positive antibody rate between the men (86.7%) and women (90.9%) for asymptomatic infection was not significantly different (p > 0.05).

| Quantitative dynamic changes in serum SARS-CoV-2 antibody in asymptomatic cases
To further detect serum SARS-CoV-2-specific IgM and IgG anti- There was no significant difference in IgM concentration between time points tested (0-7 weeks) after nucleic acid conversion (p > 0.05) (Figure 2A). However, a significant difference was observed in IgG concentrations with time changed (χ 2 = 14.034, p = 0.029) ( Figure 2B). The levels of serum IgM and IgG antibodies at different time periods (0-7 weeks) after nucleic acid negative conversion are shown in Table 2.

| DISCUSS ION
Cutting off the route of transmission is an important preventive measure for infectious diseases. However, it has been reported recently that asymptomatic carriers can lead to person-to-person transmission in a community due to neglecting, thus posing a considerable challenge for the prevention and control of COVID-19. Therefore, the proportion of asymptomatic infections needs to be determined in a timely manner through proper laboratory techniques.
There are two categories of asymptomatic patients with Remarkably, numerous studies have demonstrated that dynamically monitoring of IgM and IgG in serum was critical for COVID-19 confirmed patients, which can assist nucleic acid detection to provide valuable guidance for the diagnosis, staging, and prognosis of SARS-CoV-2 infection. 9,15 To further evaluate the production of SARS-CoV-2-specific antibodies in asymptomatic patients, blood specimens were collected regularly to detect the levels of serum SARS-CoV-2-specific IgM and IgG antibodies after negative nucleic acid conversion. The positive rate of IgM antibodies in asymptomatic individuals was significantly higher even after negative nucleic acid conversion than that in healthy people. However, there was no significant difference in changes within 7 weeks after negative nucleic acid conversion (p < 0.05). Our previous study showed that the positive rate of IgM in patients with COVID-19 was 75.9%. 16 These results suggest that IgM may be a useful target for screening cases previously infected by SARS-CoV-2 and healthy people. Furthermore, IgM in some asymptomatic individuals after negative nucleic acid conversion is not easily degraded within 7 weeks.
According to our results, the serum concentration of IgG in asymptomatic individuals after the negative nucleic acid conversion was above normal reference and increased with time in the 7 weeks of observation. The positive rate of serum IgG in the asymptomatic group was 93.5% during 7 weeks after negative nucleic acid conversion. Long et al. 17 reported that the positive rate of serum IgG in patients with acute phase COVID-19 was 18.9%, and that of patients with the convalescent-phase of COVID-19 was 60.0%. Through these data, we infer that the IgG level of asymptomatic cases increased after negative nucleic acid conversion compared with the disease and recovery period. In addition, it may also be related to our longer observation time after the nucleic acid turned negative in asymptomatic cases, when the protective antibody IgG level began to rise.
To date, studies have showed that SARS-CoV-2 IgM and IgG antibodies detection methods had high specificity and sensitivity, and combined detection was superior to individual antibody detection. Interestingly, a recent study by Hu et al. 18 found that heat-inactivated serum at 56°C for 30 min can affect the level of SARS-CoV-2 antibody in serum, which may lead to false-negative results. Additionally, Xiang et al. 19 showed that the sensitivity and specificity of IgM were 77.3% and 100%, respectively, and the sensitivity and specificity of IgG were 83.3% and 95.0%, respectively, during detection of antibodies in COVID-19 patients. Our previous study showed that the sensitivity and sensitivity of IgG (90.5% and 99.3%, respectively) were higher than those of IgM (75.9% and 94.0%, respectively) in COVID-19 patients. 16 Taken together, this indicates that the positive rate of IgM in asymptomatic patients after nucleic acid negative conversion is significantly lower than that in confirmed patients, whereas IgG remains at the same level as that of confirmed patients. Unfortunately, we were unable to obtain serum specimens from asymptomatic patients when their nucleic acid was positive, and thus it was not possible to compare changes in antibody levels between nucleic acid positive and negative.
In conclusion, nucleic acid testing, though time consuming and susceptible to sampling errors, is recommended to be the main basis for the diagnosis of asymptomatic patients. Antibody detection holds great value in the diagnosis and identification of asymptomatic patients because it is fast and convenient, and sampling can be easily standardized.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are presented in this manuscript without additional supplementary information.