key: cord-0771478-zegi0x61
authors: Liang, Lijun; Guo, Qianfang; Zhang, Huan; Lin, Shujian; Zheng, Huanyin; Li, Bosheng; Zhang, Yunqiang; Yu, Jianxiang; Zhou, Huiqiong; Liang, Yiwen; Huang, Xinxin; Wu, Jie
title: Low infectious risk of re-positive COVID-19 patients: a single-center study
date: 2021-08-12
journal: Int J Infect Dis
DOI: 10.1016/j.ijid.2021.08.019
sha: ea955d4bcab0060a34ce5b26629c3264b4872a8f
doc_id: 771478
cord_uid: zegi0x61

OBJECTIVE: This study aimed to investigate the infectiousness of re-positive COVID-19 patients. METHODS: All nucleic acid testing (NAT) was performed using throat swab, nasopharyngeal swab and anal swab were tested by real-time PCR. Re-positives were defined as a discharged patient re-tested positive in NAT. Micro-neutralization of SARS-CoV-2 was performed based on the methods of SARS and MERS viruses. IgM and IgG against N protein of SARS-CoV-2 were determined by ELISA assay. RESULTS: A total 255 (16.04%) out of 1590 COVID-19 cases were re-positive. The re-positives were more likely to occur in patients at the age of 20-39 and patients with moderate-severity. Quantitative PCR showed CT value and viral loads were both far lower than the hospitalized COVID-19 patients. The viral culture of the sample from re-positive cases showed no cytopathic effect, and NAT for the culture medium of viral culture all exhibited negative results. CONCLUSION: The viral load of the re-positive cases was very low, not infectious, and the risk of human-to-human transmission was extremely low. The discharged COVID-19 cases should undergo home health management for 3 weeks.

The coronavirus disease 2019 is caused by SARS-CoV-2 .

After the first report at the end of 2019, COVID-19 has become a global pandemic due to the high transmissibility of the virus (World Health Organization, 2020) . According to the Diagnosis and Treatment Protocol for Novel Coronavirus pneumonia (Trial Version 6) released by the China's National Health Commission (National Health Commission of China, 2020), all discharged COVID-19 patients should undergo a 14-day medical observation and quarantine. During this period, some discharged patients are found to be re-tested positive for SARS-CoV-2, which are defined as "re-positives". Several studies have reported the occurrence of re-positives in the discharged COVID-19 patients Hoang et al., 2020; Jiang et al., 2020; Peng et al., 2020; J. Yuan et al., 2020) . It has been reported that about 7-10% of COVID-19 patients are re-tested positive for SARS-CoV-2 RNA by RT-PCR after discharge (Cao et al., 2020; B. Yuan et al., 2020) . However, the infectiousness of the re-positive COVID-19 patients is largely unknown. Therefore, the current study aimed to investigate the infectiousness of re-positive COVID-19 patients. 6 

According to the Diagnosis and Treatment Protocol for Novel Coronavirus pneumonia (Trial Version 7) released by China's National Health Commission (National Health Commission of China, 2020), a patient positive for both ORF gene and N gene of SARS-CoV-2 in the nucleic acid tests was diagnosed with Covid-19. Re-positives were defined as a patient re-tested positive in nucleic acid test after reaching the discharge standard (with negative results in two consecutive nucleic acid tests). As of May-18 2020, a total of 1590 COVID-19 cases were diagnosed in the Guangdong Provincial Center for Disease Control and Prevention. Of them, 439 patients with NAbs, IgM, and IgG data were included for serological analysis, including 237 re-positive cases and 202 non-re-positive cases.

The SARS-Cov-2 nucleic acid detection kit (Shanghai Berger Medical Technology Co., China) was used to detect the ORF1ab gene and N gene of the SARS-Cov-2 virus using dual fluorescence reverse transcription-polymerase chain reaction (RT -PCR) according to manufacturer's protocol (Xu et al., 2020) . 7

The throat swabs, nasal swabs, and stool specimens of re-positive cases were collected for virus isolation in BSL-3 using Vero-E6 cells. An aliquot of 200 μL of re-positives specimens was added to a cell culture tube containing a single-layer 80-95% of Vero-E6 cells using DMEM medium (1% FCS, 100 U penicillin/mL, 100 μg streptomycin/mL, and 2 mM glutamine), followed by incubation for 3-7 days in a 37 °C, 5% CO 2 incubator. When the cytopathic effect (CPE) was observed in all cells, the supernatant was aspirated into a new culture flask for sub-culture, and part of the supernatant was stored at -80°C.

The negative COVID status was confirmed before discharge as follows: 1) Body temperature returned to normal (< 37.3℃) for more than 3 days; 2)significant relief of respiratory symptoms; 3) CT imaging showed pulmonary inflammation was obviously absorbed; 4) two consecutive NATs showed negative results, and the interval between 2 tests should be at least 1 day. For the last sample tested before discharge, throat swab, nasopharyngeal swab and anal swab were tested by real-time fluorescent quantitative PCR; 5) after discharge, the patient 8 needed to be home quarantined for 14 days and received NAT on 7 and 14 days after discharge.

Those with negative result will be released from home quarantine, and those with positive results will be hospitalized again for treatment.

For serum sample preparation, 3 mL of venous blood was collected in a vacuum blood-collection tube, followed by incubation at room temperature for 20-120 min. After that, the tube was centrifuged at 1000 rpm for 20 minutes, and the resulting supernatant was collected as a serum sample.

SARS-Cov-2 is a new virus, there has no established method of micro-neutralization test and the threshold of positive neutralizing antibody titer. Since SARS-Cov-2 belongs to β-coronavirus, therefore, the micro-neutralization test method and the threshold of positive neutralizing antibody titer were established with reference to those of SARS (Chinese Medical Association; and Chinese Society of Traditional Chinese Medicine, 2003) and MERS (GuangYu et al., 2013) . All procedures of the micro-neutralization test were 9 performed in a BSL-3 laboratory. First, 120 μL of diluted serum sample (1:4 to 1:1024 dilution), and 120 μL of SARS-CoV-2 virus stock (20SF014/vero-E6/3, titer 100TCID50/50ul) were added to the 96-well plate, followed by incubating in a 37°C 5% CO 2 incubator for 2 hours. An aliquot of 100 μL of the virus serum mixture was added to a 96-well micro cell plate containing Vero-E6 cell suspension (1×10 4~2 ×10 4 cells/0.1 mL), followed by incubating in a 37°C 5% CO 2 incubator for 5-7 days. When the 100 TCID50/50ul antigen control well shows complete cytopathic (CPE), the CPE results of each well were recorded. The reciprocal of the highest dilution of the serum that can protect 50% of the cells from CPE was defined as the titer of neutralizing antibodies for SARS-CoV-2.

The neutralizing antibody titer ≥ 1:4 was defined as a positive result.

The recombinant nucleocapsid protein (N protein)-based ELISA kit (ZhongshanShengWu, Zhongshan, China) was used for the detection of IgM (N-IgM) and IgG (N-IgG) against N protein of SARS-CoV-2 in four patients according to manufacturer's protocol. These 4 patients were originally close contacts of COVID-19 patients and then became COVID-19 patients after being infected. During the quarantine management, it was found that the 4 cases had started to produce antibodies before positive result of NAT. Since the antibody can be 10 detected at an early stage, the changes in the antibody levels can be monitored during the entire course. Briefly, serum sample (100 μL, diluted 1:100) was added to the pre-coated plates, followed by incubation with horseradish peroxidase(HRP)-conjugated rN protein of SARS-CoV-2 (100 μL, 37 o C for 30 min), and then TMB substrate solution (50 μL). The reaction was terminated by adding 50 μL of 2 M sulfuric acid, and the absorbance value at 450 nm and 630nm (A450-630) was determined. According to the instruction of the kit, the diagnostic cutoff value of IgM and IgG was 0.15 and 0.25, respectively. When A450-630 was greater than or equal to the cutoff value, the test was considered positive. Three replicates of each sample were included on each plate.

Continuous data were indicated with mean ± standard deviation (SD). For the comparisons between the two groups, the student's independent t-test was used. Mann-Whitney U test would be used if the normality of continuous variable was not assumed. Categorical variables were presented as number and percentage and were compared using the Chi-square test or Fisher's exact test (if expected value ≤ 5 was found). For the comparisons between two dependent dichotomous variables, the McNemar test was used. Logistic regression models were used to investigate the association between independent variables and re-positive. A P<0.05 would be recognized as reaching the significance of each test, two-tailed. All analyses were performed using IBM SPSS Version 25 (SPSS Statistics V25, IBM Corporation, Somers, New York).

As of May-18 2020, a total of 1590 COVID-19 cases were diagnosed in our center, and 255 (16.04%) of them were re-positive cases. The gender ratio was 1:1, and the median age was 34 years (range: 0.25 to 86). With the increase of age ( Figure 1A ) and the severity of the disease (except for critical patients, Figure 1B) , the re-positive rate decreased significantly (all P<0.05).

NAT was performed in 223 re-positive patients, and 64.13% (143/223, cumulative) occurred re-positive within 7 days after discharge, 99.55% (222/223, cumulative rate) cases were re-positive within 14 days after discharge, the range was from 1 to 64 days (median: 10). Of them, 62.78% (140/223, cumulative) cases turned negative within 2 weeks after re-positive, 71.30% (159/223, cumulative) cases turned negative within 4 weeks after re-positive. The time range of turning negative was from 6 to 75 days (median: 20). 12 Fluorescence quantitative PCR showed the mean CT value of the virus ORF1ab gene of 87 re-positive patients was 37 (range: 32 to 40), and the mean viral load was 1138 copies/mL (range: 18 to 6430), which was far lower than the hospitalized COVID-19 patients (critical, 3.0×10 7 copies/mL; sever, 1.1×10 7 copies/mL; moderate, 7.4×10 6 copies/mL; mild, 4.9×10 6 copies/mL; and no symptom patients, 6.0×10 5 copies/mL).

A total of 439 patients (mean age=40.09±19.74 years, 240 males and 199 females) with NAbs, IgM, and IgG data were included for serological analysis, including 237 re-positive cases and 202 non-re-positive cases. As indicated in Table 1 , the re-positive group had significantly less severe and critical diseases and longer sampling time after symptom onset as compared with the non-re-positive group (both P<0.05). Table 2 , the level and positive rate of IgM were significantly lower in the re-positive group than in the non-re-positive group (both P<0.05). No significance was found in NAbs and IgG results between groups.

Micro-neutralization showed positive results in 222 (93.67%) re-positive patients and 184 (91.09%) non-re-positive patients (P=0.307). For the re-positive group, a median of 58 days 13 (range: 1 to 85) after onset, the antibody could be tested positive, and the mean titer was 1:34.4 (range: negative to 1:2048).

For the non-re-positive group, a median of 42 days (range: 3 to 85) after onset, the antibody could be tested positive, and the mean titer was 1: 109.9 (range: negative to 1:1024). The titer was not significantly different between the re-positive and non-re-positive groups (P=0. 477). 

To determine viral loading, 60 respiratory and 30 gastrointestinal tract samples were collected in re-positive patients for qPCR. The mean viral load of ORF-copies/mL was 1.6×10 4 (range: 6.3×10 5 to 17×10 5 ), N-copies/mL was 1.6×10 4 (range: 6.3×10 5 to 17×10 5 ).

These results were far lower than the results before turning re-positive. 14 There were 92 re-positive patients' nasopharynx and stool samples underwent Vero-E6 cell separation in a BSL-3 lab, no cytopathic sample was found. No positive sample was detected in the qPCR of the culture medium. These might due to the low viral loads of samples ( Figure   3 ).

The "re-positive" is defined as after a COVID-19 patient recovered and discharged, SARS-Cov-2 nucleic acid can be detected again in the body. The re-positive cases have been reported in several countries, such as China (Wong et al., 2021) , Japan (Schneider et al., 2020) and Brunei (Wong et al., 2020) . In this study, our results showed that re-positives were more likely to occur in patients at the age of 20-39 and patients with moderate-severity. Continuous monitoring and sampling of re-positive patients found that 64.13% of cases were re-tested positive in nucleic acid test within 7 days after discharge, 99.55% of cases had re-positive within 14 days. Of all re-positive cases, 71.30% of cases turned negative within 4 weeks after re-positives.

For discharged patients, therefore, 14-day medical isolation and monitoring are required, and re-positive cases need to be continuously monitored for at least 14 days until turning negative.

In this study, the fluorescence qPCR detection for the ORF1ab gene showed that the mean CT value and the mean viral load were both far lower in the re-positive cases than in the 15 hospitalized COVID-19 cases. No cytopathic effect was observed during the viral culture for three passages of the re-positive cases. Furthermore, nucleic acid test all exhibited negative results for the culture medium of viral culture. These results suggested that the viral load of the re-positive cases was very low, not infectious, and the risk of human-to-human transmission was extremely low. Consistent with these findings, a Japanese study by Ogawa et al. (Schneider et al., 2020) analyzed 15 healthcare personnel who had contact exposures and aerosol exposures to re-positive COVID-19 patients, and found that all healthcare personnel were negative for SARS-CoV-2 in both serological analysis (IgG) and PCR tests (nasopharyngeal swabs) on day 10. In addition, none of them had any symptoms and apparent infection during 10 days of active isolation. Likewise, a study by Wong et al. in Brunei has reported that all 111 close contacts exposure to the 21 re-positive cases had a negative result of RT-PCR for SARS-CoV-2 (Wong et al., 2020) . All these results suggest a low infectivity potential in the re-positive COVID-19 patients.

There are still some limitations to this study. First, the sample size was relatively small. In addition, due to the retrospective nature of this study, not all patients had completed data. In the future, a large prospective trial should be conducted to validate the findings of this study. 16 In summary, this study suggested that although COVID-19 patients recovered and the viral load was too low to be isolated, SARS-CoV-2 may still be excreted at a low level. Therefore, continuous monitoring of viral load and antibody levels should be simultaneously considered to develop epidemic prevention policies. Serological analysis is especially important for patients with extremely low viral load. We recommend that discharged COVID-19 cases should undergo home health management for 3 weeks. If no symptoms are presented during the 3 weeks, the patients can return to normal life after clinical consultation. 

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This study was funded by the NIAID center of excellence for influnenza research and surveillance 

The ethical approval and the consent to participate are not required due to the retrospective nature of this study in this section.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.