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-specific antibody levels in discharged COVID-19 patients has been conducted in Wuhan city, China. The survey was performed in confirmed 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 significance, a design effect of 1.1 for cluster sampling and a 10% non-response rate, we estimated that 1047 confirmed COVID-19 cases were to be interviewed. A two-stage cluster sampling design was used to select the sample. A cluster was defined 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 confirmed COVID-19 cases were selected from the streets ranking top three in the number of confirmed COVID-19 cases in Jiangan and Qiaokou districts. All communities in the street where the number of confirmed COVID-19 cases was highest in Jiangxia district were also selected. Eventually, confirmed 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 frequency-matched by age, gender, district, and disease severity were enrolled in the study.
Procedure
The confirmed 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-specific 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 cross-sectional survey, a field 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 specific 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 Scientific, CA, USA). Cycle threshold (Ct) values <37 were defined as positive, while Ct values >40 were defined 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 defined as SARS-CoV-2 re-positive; otherwise, they were defined as SARS-CoV-2 negative.
Detection of SARS-CoV-2-specific IgM and IgG
The serum SARS-CoV-2-specific 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-specific 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-specific 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-specific 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 defined 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 cm2 areas (or all surface if areas are less than 25 cm2) 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´105 cells/well in Dulbecco modified 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-specific 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 significant.