Antibody response among COVID-19 patient with and without re-detectable positive RT-PCR results in convalescent phases

Objective: To analyze the dynamic of total, IgA, IgM and IgG antibody of the conrmed COVID-19 patients during convalescent phases to understand the kinetics of antibody response among recovered patients. Methods: From March 4 to April 29, 2020, a total of 143 recovered COVID-19 patients with clear date of illness onset available were enrolled in this study. Nasopharyngeal and anal swabs were collected for SARS-CoV-2 RNA testing. Blood samples were collected for antibodies testing. Results: A total of 275 blood samples up to 96 days after illness onset were collected from 143 recovered patients. High titers of total and IgG antibodies continued to persist for over 3 months, with 100% and 99.3% patients remaining positive for total and IgG antibody. IgM antibody declined rapidly with a median time to seronegative at 67 (95%CI: 59, 75) days after illness onset. Around 25% patients were seronegative for IgA antibody at month 3 after illness onset. No statistical signicance difference was founded in the antibody kinetics between patients with and without re-detectable positive RT-PCR results during in convalescent phases. Conclusion: Similar high antibody titers of total and IgG antibody continued to persist for over 3 months among recovered COVID-19 patients with and without re-detectable positive RT-PCR results.


Introduction
Coronavirus disease 2019 (COVID-19) is now pandemic globally which spreads rapidly to over 200 countries, with over 88 million con rmed cases and 1.9 million deaths up to 12 th January 2021 [1,2].
Some studies reported that a proportion of recovered patients had re-detectable positive of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA with real-time reverse transcriptasepolymerase chain reaction (RT-PCR) tests [3][4][5]. Young and mild COVID-19 patients seemed to have higher risk of re-detectable positive RT-PCR results during convalescent phases [3,6].The antibody level tend to be similar among re-positive and non-re-positive groups [3,6]. However, these studies included only one antibody test of each patient and did not adjusted onset time which was signi cantly correlated with antibody level. It is still unclear whether the dynamic of antibody responses in patients who have been repositive after discharge differ from those who have not.
This study retrospectively analyzed the dynamic of total, IgA, IgM and IgG antibody of the con rmed COVID-19 patients during convalescent phases to understand the kinetics of antibody response among recovered patients.

Method Study design and participants
In Shenzhen, COVID-19 patients who met the criteria of the hospital discharged were continued to be quarantined in the Shenzhen Sami Medical Center for 14 days of medical observation. The criteria for the hospital discharged including: a) normal body temperature for more than 3 days; b) signi cant improvement in respiratory symptoms; c) signi cant improvement in absorption of acute exudative lesions on lung imaging; and d) negative nucleic acid testing of two consecutive respiratory specimens (sampling interval of at least 1 day). During the 14 days quarantined period, all recovered patients would receive the SARS-CoV-2 RNA and antibody tests. After discharge from medical center, all patients were also invited to participate semimonthly or monthly follow-up to donor blood sample for antibody testing.
All participants provided written informed consents.
This study was approved by Medical Ethical Committee of Shenzhen Sami Medical Center.

Laboratory examination
Nucleic acid detection Nasopharyngeal and anal swabs were collected at 7 th and 14 th days in the medical center and tested by commercial RT-PCR assay target ORF1ab and N genes of SARS-CoV-2 as pervious described [6]. Brie y, nucleic acids were extracted by High Pure Viral RNA Kit (Roche, Mannheim, Germany). Nucleic acid ampli cation and identi cation by commercial RT-PCR test kits approved by National Medical Products Administration of China (Bio-Germ, Shanghai, China).

Antibody detection
Blood samples were collected at least one time during 14 days quarantine period in medical center, and then collected semimonthly or monthly after discharge. Total, IgA, IgM and IgG antibody against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in the sera of COVID-19 patients were tested with a commercial chemiluminescence microparticle immunoassay (CMIA, Xiamen InnoDx Biotech Co., Ltd., China) as previous describe [7]. Brie y, total antibody detection was based on the double-antigen sandwich method. The capture method was applied to detect the IgM antibody. For IgG and IgA antibody detection, indirect method was used. The test procedure of all assays were performed in accordance with the manufacturer's instructions. The antibody levels were expressed by the relative binding signals compared to the cutoff value of each assay (S/CO). The sensitivity of total, IgM, IgG and IgA antibody were 96.3%, 86.3%, 99.6% and 91.9% respectively, and the speci city of total, IgM, IgG and IgA antibody were 99.3%, 99.3%, 99.0% and 98.6%, respectively (unpublished data for IgG and IgA) [7].

Statistical analysis
Geometric mean titers (GMT) for Total, IgA, IgM and IgG antibody with 95% con dence interval were calculated by days after illness onset. Multivariable log-binomial regression models with generalized estimating equations were used to compare the antibody level between re-positive and non-re-positive groups, which adjusted for age and days after illness onset. The probability of maintaining seropositive and median time to seronegative were calculated by the Kaplan-Meier method, and compared by log-rank tests. All analyses were conducted by using SAS software (version 9.4), and p value less than 0.05 considered as statistical signi cance.

Antibody kinetics
A total of 275 blood samples were collected from 143 patients, with median 4 and 1 samples for each patient in re-positive and non-re-positive group separately. All recovered patients were seropositive for total antibody in their rst sample during the medical center. As shown in gure 1, the levels of total antibody were decreased slowly and remain at high S/CO value at around 3 months after illness onset (GMT=162.5, 95%CI: 64.7-408.0), with none patients seronegative in their last samples. For IgG antibody, the levels remain at a plateau from 1 month to 3 months after illness onset. Only one patient was negative for IgG antibody at 53 days after illness onset. The levels of IgM antibody declined fastest compared with other antibodies, with median time to seronegative at 67 (95%CI: 59, 75) days after illness onset. IgA antibody declined at a rate between IgM and IgG. Around 25% patients were seronegative for IgA antibody at month 3 after illness onset.
As shown in gure 2 and gure 3, the antibody levels and the prevalence of seropositive for total, IgG, IgM and IgA were similar among patients with and without re-detectable positive RT-PCR results (all p values higher than 0.05).

Discussion
This study shows that high antibody titers of total and IgG antibody among recovered COVID-19 patients continued to persist for over 3 months. IgM antibody declined rapidly with a median time to seronegative at around 2 months. IgA antibody declined slower than IgM, but faster than IgG. However, no statistical signi cance difference was founded in the antibody kinetics among patients with and without redetectable positive RT-PCR results during in convalescent phases.
In line with the current studies [8,9], our study con rmed that IgG antibodies remained stable over 3 months. Antibody response of SARS-CoV-2 infection may be similar to SARS-CoV-1 or other viral infection [10]. IgM antibodies response is eeting, and among half of patients, IgM antibodies would be undetectable around 2 months. Thus similar to other viral infections, IgM antibody against SARS-CoV-2 could be a marker of acute infection with SARS-CoV-2 [11].
The antibody levels and the prevalence of seropositive for total, IgG, IgM and IgA were similar among patients with and without re-detectable positive RT-PCR results. There are some preprint researches raised possibilities which thought to be related to the re-positive, such as virology, sample collecting methods and immunology. There were studies indicated that the SARS-CoV-2 nucleic acid could remain in the lower respiratory tract specimens and feces longer than respiratory tract for up to 50 days [12,5]. In addition, the samples quality and the sensitivity and speci city of the commercial detection kits may result in the false positive result which on some extent account for the happening of the re-positive patients [3]. There was a study showing that the levels of IgG and IgM were similar in the re-positive and non-re-positive patients, which was similar to this study [3]. Another preprint study suggested that the titer levels of IgG and IgM were not correlated with the clinical course, therefore the antibody levels could not be predictors of the disease process.
Some limitations should be noticed in this study. First, the convenient collection of blood samples led to unequally distributed sample sizes over time. Second, majority of patients provided only one blood sample during convalescent phases. Third, the duration of follow-up was relatively short.
In conclusion, this study con rmed that high antibody titers of total and IgG antibody among recovered COVID-19 patients continued to persist for over 3 months. The antibody response among patients with and without re-detectable positive RT-PCR results was similar during in convalescent phases.

Declarations
Funding: This research did not receive any speci c grant from funding agencies in the public, commercial, or not-for-pro t sectors.
Con icts of interest: The authors have declared that no con ict of interest exists.
Ethics approval: This study was approved by Medical Ethical Committee of Shenzhen Sami Medical Center.
Availability of data and material: The data will be available beginning 9 months and ending 36 months following article publication. The data will be shared with investigators whose proposed use of the data has been approved by an independent review committee identi ed for individual participant data metaanalysis. Proposals should be directed to yingyingsu@xmu.edu.cn or sunchangqing@ssmc-sz.com. To gain access, data requestors will need to sign a data access agreement.
Code availability: software application.