Clinical characteristics of the recovered COVID-19 patients with re-detectable positive RNA test

Clinical characteristics of the recovered COVID-19 patients with

re-detectable positive RNA test

Authors: Jianghong An,

1,*

Xuejiao Liao,

2,*

Tongyang Xiao,

2,*

Shen Qian,

2,*

Jing Yuan,

3

Haocheng Ye,

2

Furong Qi,

2

Chengguang Shen,

2

Yang Liu,

2

Lifei Wang,

4

Xiaoya

Cheng,

1

Na Li,

2

Qingxian Cai,

5

Fang Wang,

5

Jun Chen,

5

Yingxia Liu,

3

Yunfang Wang,

6

Feng Zhang,

7

Yang Fu,

8

Xiaohua Tan,

1,†

Lei Liu,

2,9,†

Zheng Zhang

2,9,†

Author Affiliations:

1

Department of Oncology and Hematology, Shenzhen Third People’s Hospital, Shenzhen

518112, Guangdong Province, China

2

Institute of Hepatology, National Clinical Research Center for Infectious Disease,

Shenzhen Third People’s Hospital, Shenzhen 518112, Guangdong Province, China

3

Department of Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen

518112, Guangdong Province, China

4

Department of Radiology, Shenzhen Third People’s Hospital, Shenzhen 518112,

Guangdong Province, China

5

Department of Hepatology, Shenzhen Third People’s Hospital, Shenzhen 518112,

Guangdong Province, China

6

Translational Research Center, Beijing Tsinghua Changgung Hospital, Tsinghua

University, Beijing 102218, Beijing , China

7

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge,

MA 02139, USA

8

School of Medicine, Southern University of Science and Technology, Shenzhen,

Guangdong, 518055, China

9

The Second Affiliated Hospital, School of Medicine, Southern University of Science and

Technology, Shenzhen 518112, Guangdong Province, China

*Contributed equally

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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

†Joint corresponding authors

Corresponding Authors:

Prof. Zheng Zhang, PhD, MD. Institute of Hepatology, Shenzhen 3rd People’s Hospital,

Shenzhen 518112, Guangdong Province, China; Phone: 86-755-81238983; Fax:

86-755-81238983; Email: zhangzheng1975@aliyun.com.

Prof. Lei Liu, MD, Shenzhen 3rd People’s Hospital, Shenzhen 518112, Guangdong

Province, China; Email: liulei332[email protected]om.

Prof. Xiaohua Tan, PhD, MD. Department of Oncology and Hematology, Shenzhen 3rd

People’s Hospital, Shenzhen 518112, Guangdong Province, China; Email:

xiaohua_t@hotmail.com.

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Summary

Background It has been reported that several cases recovered from

COVID-19 tested positive for SARS-CoV-2 after discharge (re-detectable

positive, RP), however the clinical characteristics, significance and potential

cause of RP patients remained elusive.

Methods A total of 262 COVID-19 patients were discharged from January 23

to February 25, 2020, and were enrolled for analysis of their clinical

parameters. The RP and non-RP (NRP) patients were grouped according to

the disease severity during their hospitalization period. The clinical

characterization at re-admission to the hospital was analyzed. SARS-CoV-2

RNA and plasma antibody levels were detected using high-sensitive detection

methods.

Findings Up to March 10, 2020, all of patients were followed up for at least 14

days, and 38/262 of RP patients (14.5%) were present. The RP patients were

characterized by being less than 14-years old and having mild and moderate

conditions as compared to NRP patients, while no severe patients became RP.

Retrospectively, the RP patients displayed fewer symptoms, more sustained

remission of CT imaging and earlier RNA negative-conversion but similar

plasma antibody levels during their hospitalization period as compared to

those NRP patients. When re-admitted to the hospital, these RP patients

showed no obvious clinical symptoms or disease progression indicated by

normal or improving CT imaging and inflammatory cytokine levels. All 21 close

contacts of RP patients were tested negative for SARS-CoV-2 RNA, and no

suspicious clinical symptoms were reported. However, 18/24 of RNA-negative

samples detected by the commercial kit were tested to be positive for virus

RNA using a hyper-sensitive method, suggesting the carrier status of virus

possibly existed in patients recovered from COVID-19.

Interpretation Our results showed that young and mild COVID-19 patients

seem to be RP patients after discharge, who show no obviously clinical

symptoms and disease progression upon re-admission. More sensitive RNA

perpetuity.

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detection methods are required to monitor these patients during follow-up. Our

findings provide empirical information and evidence for the effective

management of COVID-19 patients during their convalescent phase.

perpetuity.

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Introduction

Since early December of 2019 and up to March 23, 2020, over 300, 000 cases

of coronavirus disease 2019 (COVID-19) caused by novel coronavirus

(SARS-CoV-2) infection, with over 13, 000 deaths have been reported through

the world [1]. The World Health Organization has declared COVID-19 as a

pandemic [2]. Generally, the COVID-19 is less severe and less fatal than the

SARS, however, some patients, especially those who are elderly with

co-morbidities are prone to develop more severe symptoms and require

emergent medical interventions [3, 4]. Many literatures have retrospectively

analyzed the clinical characteristics of patients infected with SARS-CoV-2 [3-8].

Recently, an increasing number of patients with COVID-19 were discharged

from the hospital and received regular follow-up and observation.

Re-detectable positive (RP) of SARS-CoV-2 RNA test in some recovered

patients has been reported [9-12]. The management of RP patients has

attracted wide attention. However, the number of RP patients reported in the

literature was small, and the duration of follow-up was short. In addition, the

clinical characteristics is lacking and the potential impact and significance of

RP patients remain unknown, which makes it difficult to provide empirical

information and evidence support for the management of patients with

COVID-19 in the recovery period.

This study retrospectively analyzed the clinical characteristics of 38 RP

patients and 224 non-RP (NRP) patients recovered from COVID-19. It is found

that RP patients were characterized by younger age and milder conditions.

They also had minor symptoms, more sustained remission of CT imaging and

earlier RNA negative-conversion but similar plasma antibody levels during their

hospitalization period. They showed no obvious disease progression and

infectivity when re-admitted to the hospital. The hyper-sensitive detection

method identified SARS-CoV-2 RNA molecules from most samples that were

tested RNA-negative by the commercial kit, suggesting the carrier status of

virus possibly existed in recovering COVID-19 patients. These findings provide

perpetuity.

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key information for the effective management of COVID-19 patients during

their convalescent phase.

Methods

Study design and participants

A total of 262 confirmed COVID-19 patients discharged from Shenzhen Third

People's Hospital from January 23, 2020 to February 25, 2020 were enrolled in

this study. All discharged COVID-19 patients were continued to be isolated and

observed for 14 days, weekly followed-up and SARS-CoV-2 RNA detection

were performed timely. All discharged patients were followed-up for at least

two additional weeks after isolation. Among them, the RP patients were

re-admitted to hospital for further medical observation and close contacts were

also followed-up. The rest of the recovered NRP patients were closely

followed-up outside the hospital. This study was approved by the Ethics

Committee of The Third People's Hospital of Shenzhen (2020-115), which

waived the requirement for written patient consent for this retrospective

analysis. All patients gave their oral consent to participate in this retrospective

study.

Clinical definition

According to the guideline of the diagnosis and treatment for novel coronavirus

pneumonia (the sixth edition) published by National Health Commission of the

People’s Republic of China [13], all first diagnosis cases of COVID-19 were

confirmed according to positive respiratory RT-PCR tests. The discharge

criteria of the recovered patients included: temperature returned to normal for

more than 3 days, respiratory symptoms significantly improved, and significant

absorption of pulmonary lesions of chest CT imaging, and at least consecutive

negative RNA test results for 2 apart from each other by at least 24 hours. The

RP patients were confirmed by digestive (anal swab) and respiratory positive

RT-PCR tests. Since February 22, 2020, evaluation of negative anal swab was

perpetuity.

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supplemented for discharge criteria in Shenzhen Third People's Hospital.

Data collection

The medical records of 262 recovered COVID 19 patients including 38 RP

patients were reviewed. The epidemiological, demographic, clinical, laboratory

data of the patients were collected, summarized and analyzed. According to

the first chest CT imaging post admission, the extent of pulmonary

inflammation was divided into mild, moderate and severe condition basing on

the lesions involving unilateral lobe, multiple lobes in both lungs, and all lobes

in both lungs, respectively. According to chest CT within 7 days after admission,

the remission of the lesions was evaluated. The temporary progression was

indicated by increased lesion and persistent remission was indicated by stable

or absorbed or decreased lesions.

qRT-PCR and Sherlock assay for SARS-CoV-2 RNA detection

The quantitative reverse transcription polymerase chain reaction (qRT-PCR)

was assessed as described previously [14]. Nasopharyngeal and anal

specimens collected during hospitalization were sent to the laboratory in viral

transport case. Total nucleic acid extraction were extracted from the samples

using the QIAamp RNA Viral Kit (Qiagen, Heiden, Germany), and quantitative

RT-PCR was performed using a China Food and Drug Administration (CFDA)

approved commercial kit specific for 2019-nCoV detection (GeneoDX Co., Ltd.,

Shanghai, China) or Sherlock kit gifted from Feng Zhang lab according to the

manual. Each RT-PCR assay provided a Ct value, which is the number of

cycles required for the fluorescent signal to cross the threshold for a positive

test, a higher Ct value is correlated with a lower viral load. The specimens

were considered positive if the Ct value was ≤ 37.0, and negative if the viral

load were undetectable. Specimens with a cycle-threshold value higher than

37 were repeated. The specimen was considered positive if the repeat results

were the same as the initial result and between 37 and 40. If the repeat Ct was

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undetectable, the specimen was considered negative. All procedures involving

clinical specimens and SARS-CoV-2 were performed in a biosafety level 3

laboratory. Meanwhile, we did next-generation sequencing of samples from

three patients.

ELISA assay for anti-SARS-CoV-2 IgG and IgM antibody

Microtiter plates (Sangon Biotech) were coated overnight at 4°C with 4 μg/mL

recombinant SARS-CoV-2-RBD (Receptor binding domain) proteins (50 μL

per well) expressed by our laboratory through 293-T cells. The plates were

washed thrice with PBS containing 0.1% v/v Tween-20 (PBST) and blocked

with blocking solution (PBS containing 2% w/v non-fat dry milk) for 2 hours at

37°C. The plates were then washed with PBST. The sera were diluted to

200-fold into PBS as an initial concentration, and serial 3-fold dilutions of sera

was added to the wells and incubated at 37°C for 60 minutes. After three

washes, 100 μL of horseradish peroxidase (HRP)-conjugated goat anti-human

IgG (for IgG antibody titer detection) and IgM (for IgM antibody titer detection)

antibodies solution (Sangon Biotech) were added to each plate, respectively,

and incubated at 37°C for 60 minutes. After five washes, 100 μL of

tetramethylbenzidine (TMB) substrate (Sangon Biotech) was added at room

temperature in the dark. After 15 minutes, the reaction was stopped with a 2 M

H2SO4 solution. The absorbance was measured at 450 nm. All samples were

run in triplicate. The ELISA titers were determined by endpoint dilution.

Statistical analysis

A statistical analysis was performed using SPSS 26.0 (IBM, Chicago). All of

the statistical tests were two-sided, and significant differences were

considered at p < 0.05. Continuous variables were evaluated using the median

and interquartile range (IQR) values. Chi-square or Fisher exact tests were

utilized to compare the proportions of the categorical variables.

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Results

Demographic, epidemiological and clinical characteristics

A total of 262 patients were discharged from January 23, 2020 to February 25,

2020 and were followed-up for at least 14 days. Among them, mild, moderate

and severe patients accounted for 11.4% (n = 30), 81.0% (n = 212) and 7.6%

(n = 20), respectively. Up to March 10, 14.5% of convalescent patients (n = 38)

were re-detected to be SARS-CoV-2 RNA positive during their followed-up

period. None of severe patients were re-tested to be RNA positive

(Supplemental Table 1).

It revealed that the vast majority of RP patients (97.4%, n = 37) were

younger than 60 years of age. Among them, patients younger than 14 years

old were more common compared with those between the ages of 14 and 60

years (35.0% vs 16.0%, p < 0.01) (Table 1). In addition, it is found that 36.7%

(11/38) of RP patients are characterized by mild symptoms. The percentage

was significantly higher than what was seen among NRP patients (12.7%,

19/204, p < 0.01, Supplemental Table 1). There was no significant difference in

the gender distribution. Notably, there were less mild RP patients having fever

in their initial symptoms as compared to mild NRP patients (p < 0.01). Also,

45.5% of mild RP patients displayed only upper respiratory symptoms at the

first admission, while mild NRP patients usually had lower respiratory

symptoms at the first admission (Table 1). There is no difference of the extent

of lesions in the first chest CT imaging between RP and NRP patients with

moderate stages. However, the incidence of RP (85.2%) was found to be

particularly closely related to the sustained remission of chest CT imaging as

compared to NRP patients, of which 36.2% displayed transient progression

during their first hospitalization period (Table 1 and Figure 1). There was no

significant difference in the usage of steroid and antiviral therapy between RP

and NRP patients during their first hospitalization period. In addition, RP

patients did not show a higher incidence of a history of traveling and living in

Hubei province as compared to NRP patients.

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Differential RNA dynamic in RP patients with that in NRP patients

No differences of days of last RNA negative-conversion since the onset of

illness and hospitalization days were found between RP and NRP patients.

Importantly, RNA negative-conversion occurred mostly within 2-3 weeks since

the onset of illness among 63.6% mild RP patients and within 1-2 weeks since

the onset of illness among 22.2% moderate RP patients. By contrast, there are

more NRP patients who displayed RNA negative-conversion after 3 weeks

since the onset of illness regardless of mild or moderate status (Table 2).

These data showed RP patients were characterized by early RNA-negative

conversion while NRP patients cleared virus relatively late.

The changes of serum anti-SARS-CoV-2 IgG and IgM antibodies in

patients recovered from COVID-19

In order to evaluate the effect of serum-specific antibody levels on the

occurrence of RP, we analyzed the difference of anti-SARS-CoV-2 IgG and

IgM antibody levels in both RP and NRP patients at their discharge. More than

half of RP and NRP patients displayed medium levels of IgG and IgM

independent on their disease severity. However, there are no differences of

antibody levels in between both groups of patients (Supplemental Table 2). We

also evaluated the dynamic of IgG and IgM levels at the discharge and

re-admission in RP patients. The IgG and IgM levels were maintained at stable

levels in these RP patients during the 14 days period (data not shown).

Supplementing negative results of anal swab test at discharge failed to

reduce RP occurrence of COVID-19 patients

In order to evaluate the effects of increasing sampling site test at discharge on

RP events, we compared the occurrence events of RP before Feb 22 and after

Feb 22 after that time the negative anal swab detection was added to

discharge criterion in COVID-19 patients. Our results showed that there was

no statistical difference in the occurrence of RP patients before Feb 22 and

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after Feb 22 (14.5% vs 14.3%, p = 0.77, Figure 2). These data indicated that

supplementing detected sites of SARS-CoV-2 RNA failed to reduce the RP

occurrence in convalescent patients.

RP patients showed no obvious clinical symptoms and disease

progression.

All 38 RP patients were re-admitted to the hospital for further medical

observation. The analysis showed that all these patients had no fever. A small

number of patients reported mild cough and chest tightness, which were not

worse than before (Table 3). All patients recovered from mild conditions (n = 30)

and 37.0% from moderate patients had normal chest CT imaging without

inflammatory signs. By contrast, 63.0% (n = 17) of patients recovered from

moderate conditions had stable or reduced inflammatory signs in their chest

CT imaging (Figure 1). There were normal range of the lymphocyte count,

plasma IL-6 and CRP levels upon admission for all RP patients. Only one

patient received transient interferon-alpha inhalation therapy, and 4 patients

received low-flow oxygen inhalation therapy and traditional Chinese medicine

after admission.

In addition, because all the convalescent patients with COVID-19 in our

cohort were required to be isolated at home or under intensive isolation, only

21 close contacts were produced. Up to March 10, 2020, all of 21 close

contacts were tested to be negative for SARS-CoV-2 RNA, and no suspicious

clinical symptoms were reported in those close contacts.

Hyper-sensitive methods potentially improved SARS-CoV-2 RNA

detection in RP patients

To investigate the possible false negative due to low-sensitive commercial

RNA detection kit, we used a higher-sensitive method to detect various types

of samples from both RP patients and NRP patients with similar illness days.

For 24 samples from 15 of RP patients who were sampled after 5-7 days since

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the onset of the re-admission, 75% of spike genes and 41.6% ORF genes

could be detected to be positive using hyper-sensitive method, while only

12.5% N genes and 4.2% RF genes were detected to be positive using

commercial detection kit. Eight of fifteen RP patients were confirmed to be

RNA positive using the hyper-sensitive kit, although only 1 person was

confirmed using commercial kit. By contrasts, 8 samples from NRP patients

were detected to be negative by both methods. These data showed

hyper-sensitive methods potentially improve RNA positive detection in

samples from RP patients with negative results.

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Discussion

Several studies have shown the existence of RP patients [9-12], however their

clinical characterization as not well defined. This study retrospective analyzed

the clinical and followed-up data in a cohort of RP and NRP patients in the

same discharge period. Up to March 10, 2020, 38 RP patients were present

which accounts for 14.5% of discharged patients during the same followed-up

period. These RP patients displayed several significant features, including

younger age and mild and/or moderate symptom during their hospitalization,

which is in consistent with a previous report [9, 12]. Mild RP patients were

usually younger than 14 years old and moderate RP patients were younger

than 60 years old. By contrast, no severe patients were found to be RP

patients within the similar follow-up period. In addition, more RP patients

displayed minor symptoms in their hospitalization such as less comorbidities

and fever, and more upper respiratory symptoms. RP patients also maintained

more remission in their CT imaging than those of NRP patients. These data

indicated that RP patients were characterized by younger and minor

symptoms in their hospitalization period.

Virus load is usually thought to be related to the disease outcome [14, 15].

The present study indicated RNA negative-conversion occurred commonly 2-3

weeks since the onset of illness in moderate RP patients as compared to more

than 3 weeks in moderate NRP patients. The significantly shortened RNA

negative-conversion time may affect the persistence of high levels of adaptive

immunity [16]. Our recent studies indicated that a higher titer of antibody in the

plasma was independently associated with disease severity in patients with

COVID-19 [17]. However, RP and NRP patients displayed similar levels of IgG

and IgM in the plasma. Future study should investigate host immune

responses which were usually considered to determine the clinical outcome

especially in virus infection [18, 19].

We also comprehensively characterized the clinical symptoms of RP

patients when they were re-admitted to the hospital. No obvious clinical

perpetuity.

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evidence of disease progression or recurrence was found in these RP patients

including CT imaging and laboratory tests. And no antibiotics, steroids,

antiviral agents and continuous supplemental oxygenation were required in

these RP patients. The inflammatory response was significantly reduced.

These data indicated that the diseases of RP patients did not progress into

more severe status even if their RNA was detected to be positive for

SARS-CoV-2. More important, these RP patients have not caused new

infections after discharge. And from a recent longitudinal study in SARS-CoV-2

infected rhesus macaques, reinfection could not occur in convalescent

monkeys [20]. Long-term follow-up of these close contacts with RP patients

will warrant the evaluation of possible risk of RP.

The underlying mechanisms underlying RP occurrence remain unclear. The

possible reasons argued by a large number of experts are related to several

virological, immunological and sampling methodological factors. Virologically,

the false negatives [21], viral residual [12], intermittent viral release [12] and

viral distribution [22, 23] are usually considered to be major factors. Our data

support the notion that the false negatives using commercial kit may partially

account for the RP, because the kits had only 30%–50% positive rate of

detection [23, 24]. In 24 of various samples from RP patients, RNA was

detected to be negative for both N gene and ORF1b gene at several days after

their re-admission to the hospital using commercial kit, whose lower limit of

detection (LOD) was relatively high (500 copies/ml). However, using a more

sensitivity Sherlock kit with an LOD of 100 copies/ml [25], 75% of samples

were detected to be positive for S gene and 41.6% for ORF genes, thus

leading to half of positive subjects present within RP patients with undetectable

RNA using commercial kit in their hospitalization. By contrast, among 8

samples from NRP patients, none was detected to be positive using either

Sherlock or commercial kit. However, in a sample confirmed by SRAS-CoV-2

sequencing, the Sherlock tested it as positive (data not shown). Therefore,

future study should improve both the sensitivity and specificity of detection kit,

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which would accurately identified clinical samples. Anyway, these data

indicated that false positive detected by current kit may on some extent

account for the occurrence of RP patients.

Another virological factor is that long-term virus residual in gut and other

tissue, similar to SARS [26]. A recent study indicated that SARS-CoV-2 nucleic

acid can persist in the digestive tract and feces for nearly 50 days [27]. Thus,

extending detection time is necessary for the COVID-19 patients when they

were discharged. However, our results show that adding anal swab derived

RNA tested to be negative as discharge criterion did not significantly reduce

the occurrence of RP patients. Thus other factor may be associated with the

RP patients. We could not exclude sampling methodological factors including

differential sampling and operational methods, sample quality, and technician

expertise levels. Nor could we exclude immunological factors including low

mucosal immune responses such as low IgA levels. These factors may take

some uncertain risks leading the occurrence of RP patients [4, 27]. Future

studies should reduce RP occurrence through using hyper-sensitive detection

kit with hyper-specificity, combining detection of multiple samples with more

immune markers.

This study has several limitations. First, this study is a single-center

retrospective study and the duration of follow-up is short, and more clinical

observations are needed to evaluate the potential risk of SARS-CoV-2

recurrence and infection. Second, dynamics of SARS-CoV-2 RNA in

COVID-19 patients need to be monitored and evaluated for RP patients.

Second, additional studies should measure the dynamic changes of serum

specific antibody levels in RP patients and evaluate the continuous protective

effect of serum specific antibodies on patients with COVID-19. Finally, we

should differentiate RP patients from relapse ones from convalescent subjects,

for who two distinct prevention and control strategies will be adopted.

Taken together, our findings revealed the clinical features of RP patients

who did not show recurrence of clinical symptoms and abnormal laboratory

perpetuity.

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tests. However, hyper-sensitive detection methods revealed the existence of

SARS-CoV-2 RNA in RP patient specimens tested to be negative using the

commercial kit. Therefore, it is necessary to develop a more accurate

quantitative assessment of the RNA dynamics and additional discharge criteria

to help physicians make a decision. This study provided valuable empirical

information and clinical evidence support for effective management of

COVID-19 patients during convalescent period. Further study should evaluate

the potential clinical significance and transmission risk of RP patients.

Contributors

ZZ, LL, XT, and JA had the idea and for designed the study. ZZ, LL, XT, JA, XJ and QS

had full access to all data in the study and take responsibility for the integrity of the data

and the accuracy of the data analysis. JY, LW, XC, SQ, NL, QC, FW, JC, YL, LL and ZZ

had roles in the clinical management, patient recruitment, sample preparation and clinical

data collection. TX and CS had roles in the RNA and antibody detection experiments, data

collection and analysis. JA, HY, FQ ,YL and ZZ had roles in statistical analysis. JA, XJ, YW,

FZ, XT, LL, YL and ZZ had roles in data interpretation. JA, XL, YF, XT and ZZ wrote the

manuscript. YW, YF, XT and ZZ contributed to critical revision of the report. All authors

reviewed and approved the final version of the manuscript.

Declaration of interests

We declare no competing interests.

Acknowledgements

We acknowledge the work and contribution of all the health providers from Shenzhen

Third People's Hospital.

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Figure 1 Serial CT imaging of a representative RP and NRP patient. For

the RP patient, the first chest CT scan on admission (day 7 since the onset of

illness) showed ground-glass opacity in both lungs. At 3 day, 6 day and 10 day

after admission (day 10, day 13 and day 17 since the onset of illness), the lung

lesions on the chest CT imaging was significantly reduced accompanied by the

disappeared clinical symptoms. The patient was discharged at day 12 after

admission (day 19 since the onset of illness). At day 26 (day 33 since the onset

of illness), the patient was re-admitted without fever and cough due to positive

RNA detection. The chest CT showed no inflammatory lesions. For the NRP

patient, a chest CT scan showed a small ground glass in the upper left lung on

admission (day 3 since the onset of illness). On day 2 and 8 after admission

(day 5 and day 11 since the onset of illness), the double lower lung lesions

increased significantly on chest CT imaging although the body temperature

and the oxygenation index was returned to normal levels. On day 9, 14 and 17

after admission (day 12, day 17 and day 20 since the onset of illness), the

lesions in both lower lungs were recovered on chest CT imaging. Then the

patient was discharged without fever and cough at day 18 after admission (day

21 since the onset of illness) when SRAS-CoV-2 RNA was also detected to be

negative.

Figure 2 The number of discharge patients and RP patients each day

from Jan 23 to March 10, 2020. On Feb 22, 2020, the anal swab negative test

was added to discharge criterion. Blue, the number of discharge patients. Red,

the number of RP patients.

Research in context

Evidence before this study

Positive SARS-CoV-2 RNA test in some recovered patients has been reported, whose

management has attracted wide attention. However, the number of recovered patients

with the positive SARS-CoV-2 RNA test reported in the literature was small, and the

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duration of follow-up was short. The clinical characteristics are lacking and the

potential impact and significance of the patients remain unknown. The lack of these

data makes it difficult to provide empirical information and evidence support for the

management of patients with COVID-19 in the recovery period.

Added value of this study

First, the young and mild COVID-19 patients are prone to be tested positive for

SARS-CoV-2 after discharge. These patients display fewer symptoms, more sustained

remission of CT imaging and earlier RNA negative-conversion but similar plasma antibody

levels during the hospitalization period as compared to those NRP patients. Second,

upon re-admission, these patients show no obviously clinical symptoms and disease

progression. However, the hyper-sensitive detection method potentially recognized false

negative by the commercial kit.

Implications of all the available evidence

The current evidence strongly supports the effective management of COVID-19

patients during their convalescent phase.

cases recovered from COVID-19 tested positive for SARS-CoV-2 after

discharge (re-detectable positive, RP)

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Table 1 Baseline characteristics of enrolled patients with COVID-19

RP, re-detectable positive patients; NRP, non-re-detectable positive patients

Mild (n = 30)

Moderate (n = 212)

RP (n = 11)

NRP (n = 19)

P

value

RP (n = 27)

NRP (n = 185)

P

value

Age, median (IQR)-yr

20 (5-64)

23 (2-63)

0.98

38 (2-60)

48 (1-86)

< 0.01

> 14 and ≤ 60 years old-n.(%)

10 (90.9)

18 (94.7)

0.78

26 (96.3)

139 (75.1)

0.11

≤ 14 years old-n.(%)

4 (36.3)

6 (31.6)

0.57

3 (11.1)

6 (3.24)

0.04

> 60 years old-n.(%)

1 (9.1)

1 (5.3)

0.32

1 (3.7)

46 (24.9)

< 0.001

Gender-n.(%)

Male

4 (36.3)

10 (52.6)

0.08

12 (44.4)

90 (48.6)

0.66

Female

7 (63.7)

9 (47.4)

0.12

15 (55.6)

95 (51.4)

0.69

Comorbidities-n.(%)

1 (9.1)

0 (0)

NA

1 (3.7)

41 (22.2)

< 0.01

History of travel or residence in

Hubei-n.(%)

10 (90.9)

16 (84.2)

0.61

23 (85.2)

152 (82.2)

0.82

Fever--n.(%)

2 (18.2)

7 (36.8)

< 0.01

23 (85.2)

133 (71.9)

0.29

Upper respiratory symptoms-n.(%)

5 (45.5)

2 (10.5)

< 0.01

4 (14.8)

34 (18.4)

0.53

Lower respiratory symptoms-n.(%)

5 (45.5)

7 (36.8)

0.34

14 (51.9)

95 (51.4)

0.96

Digestive tract symptoms-n.(%)

0 (0)

2 (10.5)

NA

3 (11.1)

15 (8.11)

0.50

The lesion range of chest CT-n.(%)

Unilateral

0 (0)

NA

6 (22.2)

36 (19.4)

0.66

Multi-lobe of Bilateral

0 (0)

NA

15 (55.6)

105 (56.7)

0.92

All-lobe of Bilateral

0 (0)

NA

6 (22.2)

44 (23.7)

0.82

Chest CT imaging-n(%)

Transient progression

0 (0)

NA

4 (14.8)

67 (36.2)

< 0.05

Sustained remission

0 (0)

NA

23 (85.2)

113 (61.1)

0.05

Steroids use-n.(%)

0 (0)

NA

4 (14.8)

27 (14.6)

0.97

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Table 2 RNA detection in the enrolled patients with COVID-19

Mild (n = 30)

Moderate (n = 212)

RP (n=11)

NRP (n=19)

P value

RP (n=27)

NRP (n=185)

P Value

*Days since the onset of illness to

last RNA negative-conversion

17 (11-22)

15 (8-24)

0.71

18 (9-30)

20 (5-47)

0.17

follow-up deadline (March 10)

40 (33-47)

42 (35-49)

0.15

45 (33-54)

46 (30-72)

0.15

discharge

15 (14-22)

16 (10-23)

0.72

17 (9-29)

18 (7-35)

0.47

Days of RNA negative-conversion

since the onset of illness (n, %)

Between 7 and 14 days

3 (27.3)

8 (42.1)

0.08

6 (22.2)

18 (9.7)

0.03

Between 14 and 21 days

7 (63.6)

8 (42.1)

0.04

11 (40.7)

84 (45.4)

0.61

More than 21days

1 (9.1)

3 (15.8)

0.18

10 (37.3)

83 (44.9)

0.40

*Median (range). RP, re-detectable positive patients; NRP, non-re-detectable positive patients

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Table 3 Clinical observation of RP patients at re-admission of hospital

Mild (n = 11)

Moderate (n = 27)

Symptoms

fever

0 (0)

cough

1 (9.1)

5 (18.5)

Chest tightness

0 (0)

2 (7.4)

other

0 (0)

3 (11.1)

Chest CT imaging

Normal

11 (100)

10 (37.0)

Stable or absorb

0 (0)

17 (63.0)

Progression

0 (0)

Laboratory examination

Abnormal lymphocyte count

0 (0)

4 (14.8)

increasing serum IL-6 level

0 (0)

increasing serum CRP level

0 (0)

Treatment

Low flow oxygen

0 (0)

4 (14.8)

Traditional Chinese medicine

3 (27.3)

8 (29.6)

Antiviral therapy

0 (0)

1 (3.7)

Number of contacts with symptoms

0

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Table 4 The comparison between hyper-sensitivity and common

sensitivity detection in RP and NRP patients

Sample

Dates since the

onset of illness

Sherlock

Commercial

S gene

ORF gene

N gene

ORF1 gene

1

Anal swab

44

-

2

Nasal swab

44

+

-

3

Anal swab

44

+

-

4

Nasal swab

44

+

-

5

Anal swab

37

-

+

-

6

Nasal swab

37

+

-

7

Anal swab

42

+

-

8

Anal swab

43

-

+

-

9

Anal swab

30

+

-

10

Blood

42

+

-

11

Blood

43

-

12

Blood

30

+

-

13

Nasal swab

42

-

+

-

14

Nasal swab

43

+

-

15

Nasal swab

30

+

-

16

Anal swab

32

+

-

17

Anal swab

37

+

-

18

Anal swab

36

+

-

19

Anal swab

37

+

-

20

Anal swab

41

+

-

21

Anal swab

37

-

+

22

Anal swab

31

+

-

23

Anal swab

43

+

-

24

Nasal swab

32

+

-

total

Positive (%)

18 (75%)

10 (41.6%)

3 (12.5%)

1 (4.2%)

25

Nasal swab

47

-

26

Anal swab

47

-

27

Nasal swab

45

-

28

Nasal swab

40

-

29

Anal swab

54

-

30

Anal swab

49

-

31

Nasal swab

44

-

32

Nasal swab

48

-

total

Positive (%)

8 (0)

Note：1-24, re-detectable positive patients; 25-32: non-re-detectable positive patients.

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Figure 1

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Figure 2

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Supplemental Table 1 Analysis of disease severity in RP and NRP patients

Mild (n = 30)

Moderate (n = 212)

Severe (n = 20)

Total (n = 262)

RP

11 (36.7%)

27 (12.7%)

0 (0)

38 (14.5%)

NRP

19 (63.3%)

185 (87.3%)

20 (100%)

224 (85.5%)

RP, re-detectable positive patients; NRP, non-re-detectable positive patients

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Supplemental Table 2 Analysis of serum anti- SARS-CoV-2 IgG and IgM antibody

levels in patients with covid-19 at discharge

Mild

Moderate

RP (n = 10)

NRP (n = 10)

P value

RP (n = 21)

NRP (n = 119)

P value

Serum IgG (n, %)

High

1 (10%)

0 (0)

0.30

8 (38.1%)

41 (34.5%)

0.95

Medium

6 (60%)

4 (40.0%)

10 (47.6%)

60 (50.4%)

Low

3 (30%)

6 (60.0%)

3 (14.3%)

18 (15.1%)

Serum IgM (n, %)

High

1 (10%)

0 (0)

0.59

7 (33.3%)

39 (32.8%)

0.68

Medium

7 (70%)

8 (80.0%)

12 (57.1%)

60 (50.4%)

Low

2 20%)

2 (20.0%)

2 (9.5%)

20 (16.8%)

Note: RP, re-detectable positive patients; NRP, non-re-detectable positive patients

High, titer with more than 16400; Medium, titer between 5400 and 16400; Low, titer with less than 5400

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