Risk factors for prolonged virus shedding of respiratory tract and fecal in adults with severe acute respiratory syndrome coronavirus‐2 infection

Abstract Background The dynamic alteration and comparative study of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) RNA shedding pattern during treatment are limited. This study explores the potential risk factors influencing prolonged viral shedding in COVID‐19. Methods A total of 126 COVID‐19 patients were enrolled in this retrospective longitudinal study. A multivariate logistic regression analysis was carried out to estimate the potential risk factors. Results 38.1% (48/126) cases presented prolonged respiratory tract viral shedding, and 30 (23.8%) cases presented prolonged rectal swab viral shedding. Obesity (OR, 3.31; 95% CI, 1.08–10.09), positive rectal swab (OR, 3.43; 95% CI, 1.53–7.7), treatment by lopinavir/ritonavir with chloroquine phosphate (OR, 2.5; 95% CI, 1.04–6.03), the interval from onset to antiviral treatment more than 7 days (OR, 2.26; 95% CI, 1.04–4.93), lower CD4+ T cell (OR, 0.92; 95% CI, 0.86–0.99) and higher NK cells (OR, 1.11; 95% CI, 1.02–1.20) were significantly associated with prolonged respiratory tract viral shedding. CD3−CD56+ NK cells (OR, 0.87; 95% CI, 0.76–0.99) were related with prolonged fecal shedding. Conclusions Obesity, delayed antiviral treatment, and positive SARS‐CoV‐2 for stool were independent risk factors for prolonged SARS‐CoV‐2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with chloroquine phosphate. CD4+ T cell and NK cells were significantly associated with prolonged viral shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance.


| INTRODUC TI ON
Severe acute respiratory syndrome coronavirus 2 (SARS-  results in the coronavirus disease 2019 (COVID- 19) pandemic. 1 The clinical and epidemiological characteristics of COVID-19 and the structure of SARS-CoV-2 have been elucidated gradually. [2][3][4][5] Although people are generally susceptible to SARS-CoV-2, approximately 80% of COVID-19 are mild. 6 However, many cases were reported persistent positive for respiratory SARS-CoV-2 nucleic acid, even after acute exudative lesions in lungs were almost absorbed, and symptoms were completely relieved. 7 Some studies showed that only a small proportion of patients have gastrointestinal (GI) manifestation, not all the fecal positive patients presented GI manifestation, and SARS-CoV-2 infection and replication persisted in the GI tract even after viral RNA undetectable in the respiratory sample. 8,9 Some studies found that older age, obesity, and the lack of lopinavir/ritonavir (LPV/r) treatment were independent risk factors for prolonged respiratory viral shedding. 10,11 The relations among viral shedding duration in respiratory and fecal simultaneously, clinical parameters, and treatment efficacy of COVID-19 are still elusive.
Thus, we performed a longitudinal retrospective study of 126 hospitalized laboratory-confirmed COVID-19 patients to evaluate the risk factors associated with prolonged viral shedding, to optimize the antiviral treatment options, and to explore the relationship between the respiratory tract and fecal viral shedding duration.

| Study design and participants
This longitudinal retrospective study enrolled 126 patients who were older than 18 years old and had available SARS-CoV-2 RNA detection data in HwaMei Hospital, University of Chinese Academy of Sciences, Zhejiang, China between January 23, 2020 and April 7, 2020. Patients were diagnosed based on the diagnosis and treatment protocols from the National Health Commission of the People's Republic of China. 12 The final follow-up was up to June 15, 2020. This study was approved by the Ethics Committee of HwaMei Hospital, University of Chinese Academy of Sciences (NO. PJ-NBEY-KY-2020-008-01). Oral informed consents were obtained from all subjects. The procedures were following the ethical standards of the Helsinki Declaration.
The privacy rights of human subjects are always observed.

| Data collection
Demographic and clinical data were obtained from electronic medical records. The discharge criteria and the duration of temperature have been delineated. 7 Cases were confirmed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay for nasopharyngeal/throat swabs or sputum, and the quantification cycle (Cq) results including two targeting genes: open reading frame (ORF) 1a/1b and nuclear (N), less than 40 was defined as a positive result. 2 Respiratory tract specimens were collected and detected every other day during hospitalization, and repeat QRT-PCR testing on 3rd, 5th, 7th, 14th, 21th, and 28th days after discharge were conducted.

| Cutoff of respiratory and fecal viral shedding
The duration of viral shedding was measured as the time from symptom onset till the time when nuclei acid tested negative twice consecutively (sample collection interval of at least 1 day), without turning positive thereafter. A report by time-dependent respiratory tract viral testing among 802 initial-testing-positive patients revealed that 50% of patients turned negative in 28 days. 13

| Statistical analysis
Continuous variables were presented median and inter-quartile range (IQR), and the differences between groups were evaluated with the Mann-Whitney U test. Categorical variables were presented as frequency and percentages, and proportions for categorical variables were compared using chi-square tests or Fisher's exact test.
with prolonged viral shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance.

K E Y W O R D S
COVID-19, longitudinal study, prolonged viral shedding, risk factor, SARS-CoV-2 Univariate and adjusted multivariate logistic regression analyses were carried out to estimate the potential risk factors. Spearman's correlation analysis was performed to identify the relationship between the duration of the respiratory tract and fecal viral shedding.
All statistical analyses above were performed by IBM SPSS statistics version 24.0. (IBM, Armonk, New York, USA). The forest plot diagram was visualized using the ggplot2 and forest plot packages for R software (version 4.0.0). Two-sided p < 0.05 was defined as statistical significance.

| Laboratory findings and immunological indicators
Baseline laboratory parameters were showed in Table S1, and the T lymphocyte subsets showed that the level of NK cells in the respiratory prolonged group (>28 days) was significantly higher than those in the non-prolonged group (<28 days) on admission (p = 0.022). However, no significant difference was discov-  (Table S1).

| Treatment and outcome
All patients received antiviral therapy, and treatment was initiated at a median of 3 days (IQR, 1-7.3) from symptom onset ( Table 2) Table 2).
We identified 60 patients with both positive viral RNA tests for respiratory tract and rectal swab specimens simultaneously (Figures 1 and 2). It was worth mentioning that two patients, patient

| DISCUSS ION
The viral shedding window of COVID-19 remains largely unchar- In this study, the median duration from onset of symptoms to antiviral therapy was 3 days (IQR, 1-7.3 days), and the interval from onset to antiviral treatment more than 7 days was an independent risk factor for prolonged viral shedding. Our results were in line with the current report that timely detection and supportive care for symptomatic patients with COVID-19 contributed to the clearance of viral RNA. 15 Previous data indicated the complex interaction between obesity and increased pneumonia risk, and the underlying mechanism might involve immune system dysregulation. 16,17 A retrospective cohort study in French reported that the proportion of COVID-19 patients who required invasive mechanical ventilation increased with BMI. 18 Additionally, a previous study on influenza A H1N1 found obesity was associated with prolonged hospitalization and the need for IMV. 19 In our cohort, the obesity rate was 13.5%, and obesity was an independent predisposition factor for prolonged respiratory tract viral RNA shedding after adjustment for age and gender. Obesity-related low-grade inflammation appeared to be associated with the prognosis of virus infection, and further study is to be warranted to elucidate etiological mechanisms.
No specific antiviral drugs have been approved to be safe and effective for the treatment of COVID-19 so far. Some drugs with antiviral properties and immunomodulatory effects were officially recommended for COVID-19, including LPV/r, arbidol, CQ, etc. 12,20 LPV/r and arbidol were previously recommended for patients with SARS and MERS-CoV. 21,22 Some studies implied that the use of LPV/r and arbidol was associated with an apparent favorable clinical response to COVID-19, 23 Note: Data are presented as the median and inter-quartile range (IQR) and n (%).
p values comparing the duration of viral shedding of <28 days and >28 days are from the Mann-Whitney U test and chi-square test.

Control clarified that CQ inhibited viral replication by interfering
with SARS-CoV-2 binding to the ACE2 receptor. 26 CQ has been used to treat COVID-19 with no benefits having been observed in a multinational registry analysis. 27 Our findings demonstrated that the use of LPV/r in combination with arbidol was linked with a shorter hospital stay and duration of viral shedding compared with the use of LPV/r in combination with CQ.
T lymphocyte plays a vital role in SARS-CoV-2 elimination by stimulating antigen-(Ag) specific T lymphocytes. 28 Emerging data indicated that lymphocytopenia was one of the important predictor factors influencing exacerbation and mortality of patients with COVID-19. 29-31 Likewise, our data identified that decreased CD4+ T cell was a significant risk factor for prolonged viral shedding of the respiratory tract. NK cells were one of the predominant lymphocyte subsets and accounted for the third of intrahepatic lymphocytes. 32

Viral infection-induced NK cells present activated and mature phe-
notypes with stronger cytotoxic capabilities in a murine model. 33 However, our study showed the increased NK cell was a risk factor for prolonged viral shedding of the respiratory tract but a protective factor for fecal viral shedding. Our study also showed that 60  including T lymphocyte subsets and cytokines were not conducted in all patients, so the role of immunomodulatory effects in eliminating SARS-CoV-2 might be underestimated. Third, our study was a singlecenter study and limited by a relatively small sample size. Fourth, the underlying reason for the unparallel detection results between the respiratory tract and fecal samples needs to be clarified in the future.
In conclusion, obesity, delayed antiviral treatment, and positive SARS-CoV-2 for stool were independent risk factors for prolonged SARS-CoV-2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with CQ. Decreased CD4+ T cell and elevated CD3−CD56+ NK cells were associated with prolonged respiratory tract RNA shedding, while decreased CD3−CD56+ NK cells might be linked with shortened fecal RNA shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance.

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
Ting Cai and Liyun Fu designed the study. Shun Zhang and Hui Zhu analyzed the data and drafted the article. Honghua Ye, Yaoren Hu, Nanhong Zheng, Zuoan Huang, and Zi Xiong contributed to the acquisition of subjects and data. Hui Zhu and Liyun Fu contributed to the analysis and interpretation of data. Ting Cai has primary responsibility for the final content.

DATA AVA I L A B I L I T Y S TAT E M E N T
The original anonymized data are available on request from the corresponding author.