Re-positive discharged COVID-19 patients are at low transmission risk for SARS-CoV-2 infection– a nding from recovered COVID-19 patients in Wuhan, China

Discharged COVID-19 patients have been found to be retested positive for SARS-CoV-2 (re-positive), which has widely raised concern among the public. We investigated the prevalence and transmission risk of re-positive cases in discharged COVID-19 patients and their SARS-CoV-2-specic antibody levels in Wuhan, China. Of 1065 discharged COVID-19 patients investigated, 518 (48.64%) patients were males; the mean age was 53.29 ± 14.91 years, with a median duration of 40 (IQR: 31–47) days since discharge. 63 patients were tested re-positive for SARS-CoV-2, with the re-positive prevalence to be 5.92% (95%CI: 4.50%-7.33%). The re-positive prevalence was higher in females (7.86%, 95%CI: 5.61%-10.12%) than that in males (3.86%, 95%CI: 2.20%-5.52%, P = 0.006). Re-positive prevalence was similar in patients tested positive and negative for IgG (6.01% vs 5.56%, P = 0.821) or IgM (6.38% vs 5.07%, P = 0.394). Illness severity and duration from illness onset to retest were not associated with the risk of positive results for SARS-CoV-2 after discharge. All 196 environmental samples collected from 49 re-positive patients were tested negative for SAR-CoV-2. Only one close contact to the re-positive patient had been tested positive for SARS-CoV-2; however, he might be a previous COVID-19 case but had not been detected before. Viral culture of 6 nasopharyngeal specimens presented no cytopathic effect of Vero E6 cells. Virus sequencing of 11 nasopharyngeal specimens indicated genomic fragments of SARS-CoV-2. 898 (84.72%) patients and 705 (66.51%) patients were tested positive for SARS-CoV-2-specic IgG and IgM, respectively. Self-report symptoms at the survey were similar, regardless of the level of antibody. All the re-positive patients and their matched non-re-positive patients were tested negative for SARS-CoV-2 four months later. These ndings indicate that Testing re-positive of SARS-CoV-2 is common in discharged COVID-19 patients, but no evidence showed the transmission risk of these re-positive cases. Further isolation of recovered COVID-19 patients is unnecessary. However, only 85% recovered COVID-19 patients had SARS-CoV-2-specic antibody, which suggested discharged COVID-19 patients still had potential re-infection risk. we performed a cross-sectional survey to assess the re-positive prevalence and the SARS-CoV-2-specic antibody levels in discharged COVID-19 patients in Wuhan, China. We also investigated the transmissibility risk among the re-positive discharged COVID-19 patients. Furthermore, a follow-up investigation were conducted for the re-positive patients and non-re-positive patients frequency-matched by age, gender, district and disease severity 4 months later. The present study could provide direct evidence for the transmission risk of re-positive discharged COVID-19 patients and suggestions for the additional medical care policy for the discharged COVID-19 patients.


Introduction
Coronavirus disease 2019 (COVID- 19) is an emerging infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has been spread worldwide and caused over 36 million cases and about 1 million deaths until October 9, 2020 1 . Since COVID-19 is a novel and unprecedented communicable disease, the clinical progression, and prognosis of this disease are still poorly understood 2 . In the battle against the COVID-19 epidemic, most COVID-19 patients recovered from SARS-CoV-2 infection except for some elderly patients. To prevent the potential risk of SARS-CoV-2 transmission, COVID-19 patients need to have two negative reverse transcriptase polymerase chain reaction (RT-PCR) results for SARS-CoV-2 on consecutive samples taken at least 24 hours apart before discharge from isolation in several countries 3 . However, several studies have reported that discharged COVID-19 patients have been retested positive for SARS-CoV-2 using PCR tests [4][5][6] . An earlier study reported that 14.5% patients had positive RT-PCR results among 25 discharged COVID-19 patients 7 . A recent study reported that 3% of the 651 discharged COVID-19 patients from a hospital in Wuhan had been retested positive for SARS-CoV-2 by RT-PCR test with a median duration of 48 days since discharge 8 . These ndings suggested that positive RT-PCR results for SARS-CoV-2 seemed common in discharged COVID-19 patients, but there is still limited evidence on the re-positive prevalence of discharged COVID-19 patients at a population level.
The infectiousness of the re-positive discharged COVID-19 patients has been concerned with the increasing reports of positive cases retested among discharged COVID-19 patients. Four re-positive discharged COVID-19 health worker patients in Wuhan had not infected their family members after returning home 9 . Similar ndings from China and Korea have also indicated the low transmission risk of these re-positive COVID-19 survivors to their close contacts. Based on these ndings, WHO and CDC of several countries have updated the criteria for releasing COVID-19 patients from isolation in May, 2020 [10][11][12][13] . The updated recommendation documented that it was unnecessary to isolate COVID-19 patients after discharge, considering the laboratory supplies, equipment and personnel were limited in the widespread transmission areas. However, all of these guidelines admitted that the currently limited evidence could not ensure absolute non-infectiousness for discharged COVID-19 patients.
Another key concern for discharged COVID-19 patients is the level of the SARS-CoV-2-speci c antibody. Recovered COVID-19 patients were assumed to have antibodies to protect them against the re-infection with SARS-CoV-2. However, several recovered COVID-19 patients have been not found to develop SARS-CoV-2 speci ed antibodies 14 . Two COVID-19 cases with re-infection of SARS-CoV-2 were reported in Hong Kong and the USA by whole genome sequencing 14,15 . Interestingly, IgG against SARS-CoV-2 was undetectable after diagnosis during the second episode. Recent studies have suggested that the T-cell responses also played a key role in the protective immunity induced by SARS-CoV-2. However, the SARS-CoV-2-speci c antibody level is still one of the primary endpoints of SAS-CoV-2 vaccine development 16,17 . Therefore, it is still a crucial concern on the level of SAS-CoV-2-speci ed antibody among discharged COVID-19 patients.
In this study, we performed a cross-sectional survey to assess the re-positive prevalence and the SARS-CoV-2-speci c antibody levels in discharged COVID-19 patients in Wuhan, China. We also investigated the transmissibility risk among the re-positive discharged COVID-19 patients. Furthermore, a follow-up investigation were conducted for the re-positive patients and non-re-positive patients frequency-matched by age, gender, district and disease severity 4 months later. The present study could provide direct evidence for the transmission risk of re-positive discharged COVID-19 patients and suggestions for the additional medical care policy for the discharged COVID-19 patients.

Study setting and participants
A cross-sectional survey on the prevalence of re-positive RT-PCR test for SARS-CoV-2 and the SARS-CoV-2-speci c antibody levels in discharged COVID-19 patients has been conducted in Wuhan city, China. The survey was performed in con rmed COVID-19 cases who had already returned home after discharge from designated hospital or Fangcang shelter hospital.
To investigate the difference of prognosis between re-positive and matched non-re-positive discharged COVID-19 patients in the cross-sectional survey, a follow-up investigation was conducted on September 2-4, 2020. All re-positive discharged COVID-19 patients determined in the cross-sectional survey were frequency-matched by age, gender, district, and disease severity to non-re-positive COVID-19 patients.
This study was reviewed and approved by the institutional review board of Tongji Medical College, Huazhong University of Science and Technology (IORG0003571). Verbal informed consent was obtained from every participant.

Sample size and sampling
We calculated the sample size assuming the expected re-positive prevalence of 10% after recovery and a maximal error of 2%. Using a 5% level of signi cance, a design effect of 1.1 for cluster sampling and a 10% non-response rate, we estimated that 1047 con rmed COVID-19 cases were to be interviewed. A twostage cluster sampling design was used to select the sample. A cluster was de ned as a community. Two districts (Jiangan and Qiaokou) and the other district (Jiangxia) were randomly selected from nine districts with high prevalence and four districts with low prevalence of COVID-19, respectively. Then communities with ≥50 con rmed COVID-19 cases were selected from the streets ranking top three in the number of con rmed COVID-19 cases in Jiangan and Qiaokou districts. All communities in the street where the number of con rmed COVID-19 cases was highest in Jiangxia district were also selected. Eventually, con rmed discharged COVID-19 cases from the 8 sampled communities were enrolled in this study.
In the follow-up investigation, 65 re-positive case and 130 non-re-positive COVID-19 patients frequencymatched by age, gender, district, and disease severity were enrolled in the study.

Procedure
The con rmed COVID-19 cases were interviewed on April 1-April 15, 2020, using a questionnaire to collect their information on the demographic characteristics, date of onset, date of diagnosis, date of hospitalization, date of discharge, illness severity, self-report symptoms and medical comorbidity. A nasopharyngeal swab for each participant was collected, followed the standard protocol recommended by the Chinese Centre for Disease Control and Prevention (CDC) 18 . All nasopharyngeal swabs were maintained in the universal transport medium for later laboratory tests for detecting SARS-CoV-2. Blood samples were also collected from each participant to test the IgM and IgG levels against SARS-CoV-2.
If the discharged patients were tested positive for SARS-CoV-2, house environment sampling was performed to investigate the SARS-CoV-2 RNA in their living house. Moreover, the epidemiology investigation for their close contacts was conducted by the CDC staff members, and the nasopharyngeal swabs and blood samples were also collected from their close contacts to detect the SARS-CoV-2 RNA and SARS-CoV-2-speci c IgM and IgG. In addition, virus sequencing and viral culture had been performed to assess the transmissibility of the re-positive discharged patients.
For those re-positive cases and their matched non-re-positive discharged COVID-19 patients in the crosssectional survey, a eld investigation were performed to collect information of their health status. The nasopharyngeal swabs were also collected to detect the SARS-CoV-2 RNA with RT-PCR test.
RT-PCR test for SARS-CoV-2 RNA RNA was extracted from nasopharyngeal swabs using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA), and then were detected by reverse transcriptase polymerase chain reaction (RT-PCR) assay using the commercial kits (DAAN Gene, Guangzhou, China) for SARS-CoV-2 in a designated virology laboratory 19 . The commercial kit was used for the qualitative detection of the open reading frame 1ab (ORF 1ab) and the speci c conserved sequence of coding nucleocapsid protein N gene of SARS-CoV-2.
Primers of RT-PCR testing for SARS-CoV-2 were according to the recommendation by the Chinese CDC. All PCR tests were performed on the Applied Biosystems TM 7500 Real-time PCR System (ThermoFisher Scienti c, CA, USA). Cycle threshold (Ct) values <37 were de ned as positive, while Ct values >40 were de ned as negative for both ORF 1ab and N genes. Samples with Ct values from 37 to 40 required a second test. Discharged COVID-19 patients with positive results for both genes or two consecutive positive results for one gene were de ned as SARS-CoV-2 re-positive; otherwise, they were de ned as SARS-CoV-2 negative.

Detection of SARS-CoV-2-speci c IgM and IgG
The serum SARS-CoV-2-speci c antibodies (IgM and IgG) of the discharged COVID-19 patients were detected using magnetic chemiluminescence enzyme immunoassay (MCLIA) kits supplied by Bioscience Co. (Chongqing, China) following the manufacturer's instructions. The details for detection have been described elsewhere 20,21 . Simply, SARS-CoV-2-speci c IgM/IgG could conjugate with FITC-labeled recombinant antigens, containing the nucleoprotein and a peptide from the spike protein of SARS-CoV-2.
They could be immobilized on anti-FITC antibody-labeled magnetic particles. The interaction between SARS-CoV-2-speci c IgM/IgG and the anti-human IgM/IgG antibody conjugated to alkaline phosphatase. The chemiluminescence signals derived from alkaline phosphatase catalyzing substrate solution were used to detect the SARS-CoV-2-speci c IgM/IgG. The tests were performed on Axceed 260 (Bioscience Co., Chongqing, China), an automated magnetic chemiluminescence analyzer. The antibody level was expressed as the chemiluminescence signal value divided by the cutoff value (S/CO). IgM or IgG was de ned as positive if the S/CO value was ≥1.0; otherwise, it was regarded to be negative.

Environment sampling
For the re-positive discharged COVID-19 patients in the cross-sectional survey, their house environment sampling was performed to investigate environmental contamination and potential transmission risk Environmental samples were taken at four sites, including the surface of the bedside table, the door handle of the bathroom, the washbasin and the squatter or sitter toilet. Swabs moistened in the universal transport medium were used to collect surface samples by swabbing approximately 25 cm 2 areas (or all surface if areas are less than 25 cm 2 ) of each site. After sampling was completed, swabs were deposited in the universal transport medium for testing SARS-CoV-2.

Virus sequencing
The nasopharyngeal swabs of 11 re-positive COVID-19 patients were selected to extract total RNA using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA). Sequencing libraries were then constructed with a NEBNext Ultra II Directional RNA Kit (NEB, USA) in accordance with the manufacturer's instructions. Libraries were sequenced on a Miseq 3000 platform (Illumina) in a paired-end (150 bp) run. Reads from Illumina sequencing were assembled using Geneious (v.11.0.3) and MEGAHIT (v.1.2.9) and aligning mapped reads to the reference of SARS-CoV-2 (MN908947) for evaluating the genome coverage.
Viral culture for SARS-CoV-2 Nasopharyngeal swabs collected from 6 re-positive discharged patients were kept in sterile tubes with the universal transport medium to which DMEM containing 2.5% FBS were added. They were then centrifuged at 3000g for 15 min to remove cellular debris. Vero E6 cells were seeded on a 24-well plate at 4´10 5 cells/well in Dulbecco modi ed Eagle medium (DMEM) containing 2.5% FBS and maintained in an air-liquid interface incubated at 37°C with 5% carbon dioxide one day prior to infection. Vero E6 cells were inoculated with 250ul processed samples at 37°C for one hour, then 500ul DMEM containing 2.5% FBS was added to the cell culture. Every 3-4 days, 250ul fresh culture medium was added to the cell culture. The cells were monitored daily for cytopathic effects (CPE) by light microscopy for 6 days. Every two days, 50ul cell culture supernatant was collected for RNA extraction and SARS-CoV-2 detection using RT-PCR.

Statistical analysis
Data were presented as absolute numbers and proportions for categorical variables, and as mean ± standard deviation for continuous variables. The Chi-square test or Fisher's exact test was used to compare the re-positive prevalence of discharged COVID-19 patients in different groups. Factors associated with re-positive RT-PCR results for SARS-CoV-2 in discharged COVID-19 patients were analyzed using logistic regression, and a multivariable model was built including all variables in the univariable analyses. The Kruskal-Wallis test was used to analyze the differences in the level of SARS-CoV-2-speci c IgM and IgG among different groups. All statistical analyses were performed with SAS statistical software version 9.4 (SAS Institute Inc), and plots were draw using R Project version 3.6.1 (http://cran.r-project.org). The analyses were performed using two-sided tests. P<0.05 was considered to be statistically signi cant.
Transmissibility of re-positive discharged COVID-19 patients 196 environment samples have been collected from 49 (77.78%) re-positive discharged COVID-19 patients. All the environment samples have been tested negative for SARS-CoV-2 with RT-PCR tests. Of the 63 re-positive COVID-19 patients, 12 (19.05%) lived alone after discharge from hospital without close contacts. The other 51 re-positive COVID-19 patients had 91 close contacts, with 25 and 9 individuals had been diagnosed as con rmed COVID-19 cases and clinically COVID-19 cases during the epidemic. There was only one person tested positive for SARS-CoV-2 with RT-PCR test from the 57 close contacts to the 51 re-positive COVID-19 patients. He was also tested positive for SARS-CoV-2-speci c IgM and IgG (S/CO value for IgM, 1.22; S/CO value for IgG, 3.31) at the same time. This person had been quarantined as a close contact to a con rmed COVID-19 case (his father, a re-positive case) in February 2020. He had been tested negative of SARS-CoV-2 several times in the duration of isolation. But he reported mild diarrhea one week before the time when his father was diagnosed as a con rmed COVID-19 case. Considered his antibody level and disease history, he was inferred to be a previous COVID-19 case who had not been detected on February 2020.
After three generations of Vero E6 cells culture, indirect immuno uorescence assay presented no cytopathic effect upon infection with the nasopharyngeal specimens from 6 re-positive discharged COVID-19 cases. Nasopharyngeal swabs collected from 11 re-positive COVID-19 with different Ct values of the ORFlab gene and N gene were further used for sequencing. Except for one specimen failing sequencing, the other 10 specimens had been detected for virus fragments of SARS-CoV-2, with the coverage of the complete sequence to be 1.8%-35.8% (Table 3).

SARS-CoV-2-speci c IgG/IgM of discharge COVID-19 patients
Blood samples were obtained from 1060 (99.53%) patients to test the antibody levels against SARS-CoV-2. The mean S/CO value of SARS-CoV-2-speci c IgG in these patients was 17.00±28.04. It was higher in patients aged ≥60 years (median 9.90, IQR: 3.24-30.29) than that in those aged 40-59 years (median 5.64, IQR: 1.82-16.01) and patients aged <40 years (median 3.38, IQR: 0.78-7.85). The SARS-CoV-2speci c IgG level was similar between re-positive discharged patients and negative discharged patients (P=0.633) ( Fig. 2A). Of the 1060 patients, 898 (84.72%) patients were tested positive for SARS-CoV-2speci c IgG. Self-reported symptoms at the survey were similar in patients tested negative and positive for SARS-CoV-2-speci c IgG (Supplementary Table 5). Multivariable analyses showed that patients with old age, long interval between illness onset to diagnosis of COVID-19 and positive result for SARS-CoV-2speci c IgM had increased probability for positive result of SARS-CoV-2-speci c IgG (Supplementary Table 6).
The mean S/CO value of SARS-CoV-2-speci c IgM after recovery was 6.16±13.15. It was similar in different groups of age, illness severity and RT-PCR results for retesting SARS-CoV-2 (all P>0.05) (Fig. 2B). 705 (66.51%) of 1060 discharged patients were tested positive for SARS-CoV-2-speci c IgM. Symptoms at the survey were also similar in patients tested negative and positive for SARS-CoV-2-speci c IgM (Supplementary Table 5). Females were at lower risk for positive result for SARS-CoV-2-speci c IgM (adjusted OR, 0.74, 95%CI, 0.56-0.98). Compared to patients with <70 days from illness onset to survey retest, those with 70-77 days interval (adjusted OR, 1.47, 95%CI, 1.04-2.06) were at increased risk for testing positive for SARS-CoV-2-speci c IgM (Supplementary Table 6).

Follow-up results for the re-positive COVID-19 patients and their matched non-re-positive COVID-19 patients
Total 48 re-positive cases and 90 matched non-re-positive recovered COVID-19 patients had been investigated on September 2-4, 2020, all of them were negative for SARS-CoV-2 with RT-PCR test. The self-report symptoms of the re-positive cases and 90 matched non-re-positive recovered COVID-19 patients at the investigation were similar (Supplementary Table 7).

Discussion
In the present study, we have reported the re-positive prevalence for SARS-CoV-2 in discharged COVID-19 patients in Wuhan, China. We found that 5.92% discharged COVID-19 patients in communities had been tested re-positive for SARS-CoV-2 with a median duration of 40 days since discharge. The females and younger discharged COVID-19 patients had higher re-positive prevalence than males and older did. The duration from onset to retest or illness severity was not associated with the risk of re-positive results for SARS-CoV-2 after discharge. The results of viral culture, virus sequencing, environment sampling and investigation of close contacts suggested there was no transmission risk of these re-positive discharged COVID-19 patients. The SARS-CoV-2-speci c IgM and IgG levels had not re ected the status of viral RNA and self-reported symptoms. Only about 85% people have IgG antibody after discharge. The follow-up investigation suggested that there were no difference for the prognosis of re-positive cases and the matched non-re-positive cases four months later.
Our study suggested that the re-positive result for SARS-CoV-2 was common among discharged COVID-19 patients. Several large hospital-based studies reported that the proportion of testing re-positive in discharged COVID-19 patients was about 3% to 10.0% depended on the days since discharge 8,22 . Our study found that females and young people had higher re-positive prevalence in discharged COVID-19 patients in Wuhan. However, one study in Guangzhou (outside of Wuhan) demonstrated that only the duration of viral shedding and Ct value were associated with retest positive event 23 . These studies have not found the association between the severity of COVID-19 and the risk of retest positive.
Currently, the mechanisms on re-positive results for SARS-CoV-2 in discharged COVID-19 patients has not been well established. First, the RT-PCR test might be false negative just before discharge. The RT-PCR sensitivity for nasopharyngeal swab in COVID-19 patients was only 38% according to the early study in Wuhan 24 . Later studies also reported similar ndings 25,26 . Second, PCR test re-positive suggested these discharged patients were still in recovery rather than re-infection or recurrence. Studies on re-positive COVID-19 patients demonstrated that these patients had improved clinical symptoms and indicators compared with that during their rst hospitalization 4,27 . Third, current evidence did not support the discharged COVID-19 patients are at risk for "re-infection". An animal model study documented Rhesus macaques infected with SARS-CoV-2 after recovery from initial SARS-CoV-2 infection did not show any viral replication in all primary tissues, clinical symptoms and histopathological changes 28 . Further longtime follow-up studies are needed to illustrate the underlying mechanisms of test re-positive.
Since re-positive test for SARS-CoV-2 was common in discharged COVID-19 patients, it raised questions on the infectivity of the re-positive discharged COVID-19 patients. Evidence from our study suggested that the transmission risk of these re-positive discharged COVID-19 patients was low. Detection of viral RNA in re-positive COVID-19 patients did not necessarily mean that they were infectious and able to transmit the virus to close contacts. A study has studied the relationship between cycle threshold (Ct) values for SARS-CoV-2 with RT-PCR test and the infectivity of respiratory samples and found that the SARS-CoV-2 infectivity to Vero cell lines was only observed from RT-PCR con rmed positive samples with Ct < 24 29 .
The Ct values of all re-positive nasopharyngeal samples in our study were high than 24 and the cytopathic effect was not observed in Vero cell culture. Furthermore, we found that self-reported symptoms were not different between re-positive discharged patients and negative discharged patients. One study has investigated chest CT ndings from re-positive discharged COVID-19 patients and found no speci c clinical symptoms of them 30 . All of these indicated that re-positive discharged COVID-19 patients in Wuhan might have no transmission risk to others. Our study showed that the level of antibodies against SARS-CoV-2 was not associated with the status of viral RNA tests in discharged COVID-19 patients. It suggested that serology tests for discharged COVID-19 patients could not identify the patients is free of SARS-CoV-2 infection or not when molecular testing was not available. According to recommendation 31 , the serology test seems to play a different role in the diagnosis and recovery stage in COVID-19 development. The value of the serology test for recovered COVID-19 still needs more evidence. According to the ndings from T cell responses to SARS-CoV-2 in Humans, CD4 + T cell responses to spike of the virus were correlated with the magnitude of the anti-SARS-CoV-2 IgG titers 32 . The IgG antibody level has also been found to be predictive of in vitro neutralizing capacity 33 . All these evidence suggested that the recovered COVID-19 patients with IgG antibody may have both neutralizing antibody and T-cell responses against the virus re-infection. But it is still unknown whether the recovered people without detectable IgG antibody face the risk of SARS-CoV-2 re-infection.
Based on our ndings from discharged COVID-19 patients in Wuhan, here are some recommendations for COVID-19 prevention. First, the re-positive cases have low transmission risk for SARS-CoV-2 spread. There is no need to isolate recovered COVID-19 patients for 14 days after discharge. However, if a discharged COVID-19 patient has new onset symptoms, he/she may suffer the COVID-19 recurrence. Such recurrent COVID-19 case may cause potential community outbreak. Considering this point, it is necessary to followup and conduct surveillance on the health of discharge COVID-19 patients. Second, for the screening and management of imported COVID-19 cases, it is unnecessary to isolate all passengers with RT-PCR test positive for SARS-CoV-2. If a passenger has no symptom but positive RT-PCR test and positive IgG antibody, he/she is referred to a recovered COVID-19 patient and do not need quarantine or isolation. It will help retard the medical overwhelmed and save limited medical resources in COVID-19 epidemic areas. Third, it is common that the recovered COVID-19 patients are tested negative for SARS-CoV-2speci c IgG. That means the recovered COVID-19 patients also need to take steps to prevent getting COVID-19. More evidence are needed for the re-infection risk of recovered COVID-19 patients.
Although it is the rst population-based study on the re-positive prevalence for SARS-CoV-2 among the COVID-19 epidemic center in China, there are still some limitations in the present study. First, cluster sampling was used to recruit discharged COVID-19 patients from communities. The age and gender of the participants were similar to that of the COVID-19 cases in Wuhan, but the proportion of severe/critical patients were smaller in this study. The enrolled subject may not be a well representative sample of recovered COVID-19 patients in Wuhan. Second, we identi ed the SAS-CoV-2 RNA by nasopharyngeal swabs, but studies have demonstrated that the positive ndings from saliva or rectal swabs from recovered COVID-19 patients were higher. The false-negative PCR test from nasopharyngeal swabs might not re ect the infectious status of discharged COVID-19 patients. Third, the present study is a crosssectional study. The associations between the related factors and the status of viral RNA, antibody might not be causal. The prospective study is still on going to validate our ndings.
In conclusion, this study suggested that testing re-positive for SARS-CoV-2 was common in discharged COVID-19 patients, but no evidence showed their transmission risk. However, not all recovered COVID-19 patients have SARS-CoV-2-speci c IgG antibody, which suggested the potential re-infection risk of discharged COVID-19 patients.

Competing interests
The views expressed in this study are those of the authors and do not represent the o cial position of their organizations. All of authors declare no competing interests.
Author contributions SW and JH had designed the study and had full access to all of the data in the study and take  * The Pearson correlation coefficients of days from onset to retest and days from onset to diagnosis, days from onset to hospitalization were 0.50 and 0.46, respectively. Therefore, only days from onset to retest were included in the logistic analyses. Table 3. Viral sequencing result of the nasopharyngeal specimens obtained from 11 re-positive discharged COVID-19 patients