key: cord-0929382-6pui91uu
authors: Gao, Chun; Zhu, Li; Jin, Cheng Cheng; Tong, Yi Xin; Xiao, Ai Tang; Zhang, Sheng
title: Proinflammatory cytokines are associated with prolonged viral RNA shedding in COVID-19 patients
date: 2020-10-14
journal: Clin Immunol
DOI: 10.1016/j.clim.2020.108611
sha: bdb87009f63c623f397610982641c32904a2fbbd
doc_id: 929382
cord_uid: 6pui91uu

Since December 2019, Coronavirus Disease 2019 (COVID-19) has emerged as a global pandemic. We aimed to investigate the clinical characteristics and analyzed the risk factors for prolonged viral RNA shedding. We retrospectively collected data from 112 hospitalized COVID-19 patients in a single center in Wuhan, China. Factors associated with prolonged viral RNA shedding (≥28 days) were investigated. Forty-nine (43.8%) patients had prolonged viral RNA shedding. Patients with prolonged viral shedding were older and had a higher rate of hypertension. Proinflammatory cytokines, including interleukin-2R (IL-2R) and tumor necrosis factor-α (TNF-α), were significantly elevated in patients with prolonged viral shedding. Multivariate analysis revealed that hypertension, older age, lymphopenia and elevated serum IL-2R were independent risk factors for prolonged viral shedding. This comprehensive investigation revealed the distinct characteristics between patients with or without prolonged viral RNA shedding. Hypertension, older age, lymphopenia and high levels of proinflammatory cytokines may be correlated with prolonged viral shedding.

The entire world has been threatened by the outbreak of coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). 1-2 As of May 5 th , 2020, over 3,500,000 cases were confirmed worldwide with a rough death rate of approximately 5.5%.

Clinical manifestations of COVID-19 are variable, and they vary from asymptomatic to mild, moderate, severe and critical. In most cases, COVID-19 patients present with symptoms including fever, cough, dyspnea, chest tightness, fatigue, gastrointestinal symptoms, myalgia and headache. Laboratory findings featuring lymphopenia and radiographic findings of pneumonia are common evidence of the diagnosis. Severe or critical patients might suffer from acute respiratory failure, acute respiratory distress syndrome (ARDS) and other serious complications involving multiple systems. [3] [4] Real-time reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 is the standard laboratory examination to confirm the diagnosis of COVID- 19. 5 Many studies have found that older patients have a higher risk of having a severe case and they have a higher death rate from COVID-19, which may be related to a weaker host immune response for antiviral defense and an uncontrolled proinflammatory cytokine storm. 3, 4, 6 A recent report by Qin et al. showed that severe J o u r n a l P r e -p r o o f Journal Pre-proof cases of COVID-19 had a profound dysregulation of their immune response. This might be part of the pathological process of COVID-19 and be related to the clinical outcome. 7 Viral shedding patterns of SARS-CoV-2 have been revealed in several studies, and the dynamics of viral shedding have been reported. 9 According to recent reports of the SARS-CoV-2 viral nucleic acid shedding pattern, a certain proportion of patients may experience a longer duration of viral nucleic acid negative conversion. [10] [11] Therefore, this retrospective study aimed to analyze the clinical characteristics of COVID-19 patients who experienced prolonged viral shedding and to investigate the contributing risk factors.

We retrospectively enrolled 112 hospitalized patients ( 

Data were collected from the electronic medical record system. The following information was collected for analysis: 1. Demographic characteristics such as age, sex, J o u r n a l P r e -p r o o f 

Throat swab samples or deep nasal cavity swab samples were collected for extracting RNA to confirm the diagnosis of COVID-19 infection as previously reported. 11 The collected swabs were placed into a collection tube with 200 μL of virus preservation solution, and total RNA was extracted within 2 hours using magnetic beads (Tianlong, Xi'an, China). The extraction solution was used for a one-step RT-PCR assay of 

We present the continuous variables as the mean ± standard error of the mean (SEM) or median (interquartile range, IQR) and analyzed them with the chi-square test or Mann-Whitney U test. We report the categorical variables as whole numbers and percentages.

Univariate logistic regression was used to evaluate potential risk factors for prolonged viral shedding. Only factors with a p-value<0.1 in univariate analysis were included in the final multivariate analysis model. We employed multivariate Cox regression to identify independent predictive factors for the length of viral RNA shedding. All p values were reported as two-sided with a significance level of 0.05. All statistical tests were calculated using SPSS version 24.0 (IBM, NY, USA).

We included 112 patients diagnosed with COVID-19 in this study. The majority of patients (94.6%) were classified as moderate. No patient was transferred to the ICU or 

The baseline laboratory and radiological characteristics of patients with and without prolonged viral RNA shedding are shown in Table 2 . Compared with the nonprolonged group, patients with prolonged viral shedding had a significantly lower lymphocyte count (0.9×10 9 /L vs 1.1×10 9 /L, p<0.001) and a higher neutrophil count (4.3×10 9 /L vs 3.3×10 9 /L, p<0.001). Prolonged cases showed significantly elevated serum infection markers, such as C-reactive protein (CRP) and ferritin (p<0.001). Levels of proinflammatory cytokines, such as interleukin (IL)-2R (p<0.001) and tumor necrosis factor-α (TNFα) (p<0.001), were significantly higher in patients in the prolonged group.

Moreover, serum albumin was lower in the prolonged group (p<0.001). On admission, the proportion of bilateral pneumonia was similar between the two groups (p=0.181).

In the multivariate survival analysis, we identified that age (≥65 yrs vs. <65 yrs), preoperative lymphocyte count, serum interleukin-2R and hypertension were independent risk factors associated with prolonged viral RNA shedding in COVID-19 J o u r n a l P r e -p r o o f Table 3) .

We investigated the differences in treatments between patients with nonprolonged and prolonged viral shedding. As shown in Table 4 receiving immunoglobulin and thymalfasin, respectively. Compared with the nonprolonged group, patients with prolonged viral shedding were more likely to receive corticosteroids (p=0.022) and hydroxychloroquine (p=0.007). The differences in the other treatments between the two groups were not statistically significant (p>0.05). In addition, we also analyzed the influence of different therapies on laboratory characteristics such as lymphocytes and proinflammatory factors (Supplemental The dynamics of the proinflammatory cytokines between the prolonged and nonprolonged groups demonstrated distinct patterns (Figure 2) . The pooled mean values of IL-2R and TNF-α were higher with a declining trend during the observation period in patients with prolonged viral shedding ( Figure 2A&B ). IL-1β and IL-8 showed a fluctuation curve from week 1 to week 7, and the differences between the two groups were not significant. IL-6 was significantly higher in the prolonged group and continued to decline until week 4, with a slightly increasing trend thereafter. Details of the pooled mean values of proinflammatory cytokines are shown in Supplemental Table 2 .

The fast transmitted COVID-19 has emerged as a global pandemic and has challenged the healthcare system all over the world. Until now, no specific treatment has been validated for COVID-19. The key to limiting virus spread is the diagnosis and quarantine of infected cases. This is a comprehensive report of 112 COVID-19 patients investigating the distinct characteristics and risk factors for prolonged SARS-CoV-2 viral RNA shedding.

In our study, we found that COVID-19 patients with prolonged viral shedding were older (p<0.001) and presented with a higher rate of hypertension (p<0.001).

Symptoms such as expectoration, breath shortness, fatigue and chest distress were more frequent in patients with prolonged viral shedding. Previous studies have suggested that patients. 21 Together with the above evidence, we showed for the first time that elevated levels of proinflammatory cytokines, such as IL2R, IL-6 and TNFα, may correlate with prolonged viral shedding. Prolonged viral shedding in certain cases may arise when the host antiviral defense and immune system are not strong enough to completely block virus replication.

In our study, we found that a higher proportion of patients in the nonprolonged group received immunoglobulin (34.9% vs. 26.5%). Although the difference is not significant, it might provide a clue for the application of immune enhancers in the early stage of disease in selected patients. 22 In addition, inhibitors such as ruxolitinib that block the JAK1/JAK2 signal transduction pathway induced by pro-inflammatory cytokines may be promising treatments for prolonged viral RNA shedding. 23 Various reports have revealed the phenomenon of SARS-CoV-2 "recurrence" or "repositive". 24, 25 Current guidelines suggest that two consecutive negative RT-PCR test results are one of the criteria for discharge. We argued that due to the high false negative rate of the viral test and an underestimated proportion of patients with prolonged viral shedding, the above patients may experience prolonged viral shedding rather than "recurrence". 26 According to our results, we recommend actively monitoring proinflammatory cytokines in selected COVID-19 patients with a high risk of prolonged viral shedding.

The world is now facing a pandemic caused by SARS-CoV-2. This study focused on investigating the risk factors for COVID-19 patients with prolonged viral shedding.

We also monitored the dynamics of proinflammatory cytokines in patients with prolonged viral shedding. The present study has several limitations that should be taken J o u r n a l P r e -p r o o f into consideration. First, in this retrospective setting, some data were incomplete.

Second, the treatments varied among patients. In clinical practice, patients would not receive antiviral treatment when they were first admitted to the hospital. Tentative antiviral treatments such as hydroxychloroquine or lopinavir and ritonavir were prescribed in patients with persistent detection of SARS-CoV-2 by RT-PCR. Therefore, it is difficult to analyze the effects of remaining positive, which might bias the results and not reflect the results of treatment.

In summary, in this study, we investigated the distinct dynamics of the inflammatory response in COVID-19 patients with prolonged viral RNA shedding. Hypertension, older age, lymphopenia and high levels of proinflammatory cytokines were correlated with prolonged viral shedding. We suggest longer observation and monitoring of serum proinflammatory cytokines in selected high-risk patients.

All authors declare that there are no conflicts of interest.

This study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All procedures followed in this study were in accordance with the 1964 Helsinki Declaration and later versions.

Written informed consent was waived by the Ethics Commission of the designated hospital for emerging infectious diseases.

The database used and/or analyzed during the current study is not publicly available (to maintain privacy) but can be obtained from the corresponding author on reasonable request.

Zhang Table 2 . Laboratory and radiological findings of patients with COVID-19 Table 3 . Multivariate analysis of risk factors for prolonged viral shedding Supplemental Table 2 TNFα, pg/mL 0-8.1 8.6 (6.9-10.5) 8.0 (6.5-9.1) 9.9 (7.2-11.9) <0.001 J o u r n a l P r e -p r o o f

Outbreak of pneumonia of unknown etiology in Wuhan China: the mystery and the miracle

Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: Summary of a report of 72314 cases from the Chinese Center for Disease Control and Prevention

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia inWuhan, China: a . Lancet

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Expression of elevated levels of proinflammatory cytokines in SARS-CoV-infected ACE2+ cells in SARS patients: relation to the acute lung injury and pathogenesis of SARS

Dysregulation of immune response in patients with COVID-19 in Wuhan, China. Clinical Infectious Diseases

SARS-CoV-2 viral load in upper respiratory specimens of infected patients

Factors associated with negative conversion of viral RNA in patients hospitalized with COVID-19

Dynamic profile of RT-PCR findings from 301 COVID-19 patients in Wuhan, China: A descriptive study

Interim Infection Prevention and Control Recommendations for Patients with Confirmed Coronavirus Disease 2019 (COVID-19) or Persons Under Investigation for COVID-19 in Healthcare Settings

China National Health Commission. Diagnosis and treatment of 2019-nCoV pneumonia in China. (Version 5) In Chinese

Clinical Characteristics of Coronavirus Disease 2019 in China

Clinical feature of COVID-19 in elderly patients: a comparison with young and middle-aged patients

Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome

Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology

Long-term infection of SARSCoV-2 changed the body's immune status

Viruses and the diversity of cell death

Expression of elevated levels of pro-inflammatory cytokines in SARS-CoV-infected ACE2+ cells in SARS patients: relation to the acute lung injury and pathogenesis of SARS

Pathogenic T cells and inflammatory monocytes incite inflammatory storm in severe COVID-19 patients

High-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with Coronavirus Disease 2019. Open Forum Infectious Diseases

Inhibition of cytokine signaling by ruxolitinib and implications for COVID-19 treatment

Positive RT-PCR test results in patients recovered from COVID-19

The authors declare that they have no competing interests and no financial support to declare. We thank Ms. Cheng Chen for English grammar correction of this manuscript.

All authors participated in the study design. Sheng Zhang: Conceptualization, Writing