key: cord-333429-bq7kfpby
authors: Shi, Ding; Wu, Wenrui; Wang, Qing; Xu, Kaijin; Xie, Jiaojiao; Wu, Jingjing; Lv, Longxian; Sheng, Jifang; Guo, Jing; Wang, Kaicen; Fang, Daiqiong; Li, Yating; Li, Lanjuan
title: Clinical characteristics and factors associated with long-term viral excretion in patients with SARS-CoV-2 infection: a single center 28-day study
date: 2020-07-02
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa388
sha: 
doc_id: 333429
cord_uid: bq7kfpby

BACKGROUND: Despite the ongoing spread of COVID-19, knowledge about factors affecting prolonged viral excretion is limited. METHODS: In this study, we retrospectively collected data from 99 hospitalized patients with COVID-19 between January 19 and February 17 in Zhejiang Province, China. We classified them into two groups based on whether the virus test results eventually became negative. Cox proportional hazards regression was used to evaluate factors associated with SARS-CoV-2 shedding. RESULTS: Among 99 patients, 61 patients had SARS-CoV-2 clearance (virus-negative group), but 38 patients had sustained positive results (virus-positive group). The median duration of SARS-CoV-2 excretion was 15 days (IQR 12-19) among the virus-negative patients. The shedding time was significantly increased if fecal SARS-CoV-2 RNA test results was positive. Male sex (HR, 0.58 [95% CI, 0.35-0.98]), immunoglobulin use (HR, 0.42 [95% CI, 0.24-0.76]), APACHE II score (HR, 0.89 [95% CI, 0.84-0.96]), and lymphocyte count (HR, 1.81 [95% CI, 1.05-3.1]) were independent factors associated with a prolonged duration of SARS-CoV-2 shedding. Antiviral therapy and corticosteroid treatment were not independent factors. CONCLUSIONS: SARS-CoV-2 RNA clearance time was associated with sex, disease severity and lymphocyte function. The current antiviral protocol and low-to-moderate dosage of corticosteroid had little effect on the duration of viral excretion.

A c c e p t e d M a n u s c r i p t for asymptomatic patients. Shedding cessation was defined as the occurrence of two consecutive RT-PCR negative results of respiratory specimens in a 24-hour interval.

For most variables, descriptive statistics such as the median with interquartile range (IQR; for data with skewed distribution) and proportion (%) were calculated. The T test, Mann-Whitney U test, and Kruskal-Wallis test were used for continuous variables. The χ 2 test and Fisher's exact test were used for categorical variables. To identify risk factors associated with a prolonged duration of SARS-CoV-2 RNA shedding, we performed a time-dependent Cox proportional hazards model adjusted for baseline covariates.

Outcomes were defined as time to SARS-CoV-2 RNA negativity, and we censored patients if they never cleared SARS-CoV-2 RNA during hospitalization. Kaplan-Meier curves were used to estimate the cumulative SARS-CoV-2 RNA negativity rate and the stratified log-rank statistic to compare the differences in viral clearance between the two groups. Statistical analyses were performed using SPSS software, version 20.0. For all analyses, probabilities were two-tailed, and a two-tailed P value of < 0.05 was considered significant. ; P < 0.01). The number of elderly patients (>65 years old) in the virus-positive group was significantly higher than that in the virus-negative group (P < 0.01). A total of 61.6% of patients were male, but no significant difference in sex was found between the two groups. Hypertension (n=36, 36.4%) and diabetes mellitus (n=16,16.2%) were the most common comorbidities among the COVID-19 patients. Additionally, a few patients had chronic lung disease, cardiac disease, immunosuppressive disease and pregnancy, but there were no significant differences between the two groups (Table 1) .

Among laboratory indicators tested at admission, inflammatory indexes such as CRP (Table 1) . Other laboratory indicators, such as leukocyte count, hemoglobin, platelet count, coagulation profile, creatine, liver function, and A c c e p t e d M a n u s c r i p t myocardial enzyme showed no difference between the two groups (Supplementary Table 1 ). From the results, persistent virus-positive patients showed lower immune cell counts and higher inflammation levels.

Only 1 patient (1.6%) tested SARS-CoV-2 negative within 5 days, 9 patients (14.7%) tested negative within 10 days, and 49 (80.3%) tested negative within 20 days from illness onset. In addition, a small subset of 12 patients with SARS-CoV-2 had detectable levels of the virus up to 30 days from symptom onset. The median time of persistent viral shedding was 16 days (IQR [13] [14] [15] [16] [17] [18] [19] [20] [21] . The virus-positive group showed a median viral shedding time of 19 days, which was significantly longer than that in the virusnegative group (median 15, IQR 12-19, P =0.002, Table 2 ). No apparent difference in time from illness onset to COVID-19 diagnosis was found between the two groups.

SARS-CoV-2 RNA was positively detected in the stool specimens of 21 patients Table 2 ). Darunavir (800 mg once daily) was prescribed to replace LPV/RTV if patients experienced significant drug side effects. The duration from illness onset to ARV start was 6 days (IQR 4-8.5). There was no significant difference in ARV start time between the virus-positive and virus-negative groups. A total of 77 patients (77.8%) received corticosteroid treatment with an initial dosage of 60 mg/d (IQR 40-80), and the time from illness onset to corticosteroid treatment was 8 days (IQR 6-10). There was no significant difference in the application, initial dosage or start time of corticosteroid treatment between the two groups ( Table 2) . Forty-nine patients (49.5%) were treated with antibiotics, and the median time from illness onset to antibiotic use was 9 days (IQR 6-13). There was no difference in antibiotic use proportion or antibiotic start time between the two groups (P>0.05). In addition, 43 patients (43.4%) received intravenous immunoglobulin treatment. The proportion of patients receiving immunoglobulin therapy in the virus-positive group was much higher than that in the virus-negative group (63.2% vs 31.1%, P=0.002).

The patients with persistent SARS-CoV-2 RNA positivity showed greater disease severity ( Table 2) . Among 99 COVID-19 patients, a total of 30 (30.3%) were transferred to the intensive care unit (ICU), and the patients in the virus-positive group had a higher rate of ICU admission than the virus-negative group (52.6% vs. 16 .4%, A c c e p t e d M a n u s c r i p t P<0.01). The median length of ICU stay for the 30 patients was 7.5 days (IQR 4-11), and in the virus-positive group, the length of ICU stay was 8.5 days (IQR 6.3-11), which was longer than that in the virus-negative group (4 days, IQR 3-5.8, P <0.01). The Acute Physiology and Chronic Health Evaluation II (APACHE II) score was used to evaluate the severity of hospitalized patients as well as to predict mortality [10] . The APACHE II score was 9.5 (IQR 5-15) in virus-positive group, which was much higher than that of virus-negative patients, with a score of 5 (IQR 2-8). Mechanical ventilation was performed in a minority of patients (12.1%). Compared with the virus-negative group, the virus-positive group had a higher rate of mechanical ventilation (26.3% vs.

3.3%, P<0.01). Moreover, all patients receiving extracorporeal membrane oxygenation (ECMO) treatment were from the virus-positive group (15.8% vs. 0%, P<0.01), indicating more severe lung injury and oxygenation.

We conducted a multivariable time-dependent Cox proportional hazards model to identify risk factors associated with the duration of SARS-CoV-2 RNA shedding ( (Table 3) . Interestingly, the use of immunoglobulin may prolong the duration of viral shedding. We found that the disease tended to be more severe in patients prescribed immunoglobulins, and these patients were older with higher APACHE II scores and worse immune function than those who did not use immunoglobulins (Supplementary Table 2 ).

Using Kaplan-Meier survival analysis, we found SARS-CoV-2 RNA clearance was associated with disease severity, which was significantly delayed in patients who were transferred to ICU and whose APACHE II scores >10 compared with that of patients in the general ward and whose APACHE II scores ≤ 10 (log-rank test, P<0.001; Figure   1A , 1B). Compared with patients with a high lymphocyte count (> 0.5×10 9 /L), patients with low immunity (lymphocyte count ≤ 0.5×10 9 /L) showed a significant delay in virus clearance (log-rank test, P=0.002; Figure 1C ). It took longer for fecal viral RNApositive patients to clear SARS-CoV-2 RNA than fecal viral RNA-negative patients (log-rank test, P=0.003; Figure 1D ).

Despite the ongoing spread of COVID-19, knowledge of the factors affecting prolonged viral shedding is still limited. Thus, we conducted a retrospective, single-center study of 99 hospitalized patients with confirmed SARS-CoV-2 infection identified between January 19 and February 17, 2020 in Zhejiang Province, China. We identified that male sex, immunoglobulin use, APACHE II score, and lymphopenia were independent risk factors associated with the duration of SARS-CoV-2 RNA shedding, whereas ARV A c c e p t e d M a n u s c r i p t combination therapy and corticosteroid treatment were not independent factors. In addition, patients with fecal viral RNA tested positive needed more time to clear SARS-

Consistent with previous findings, median age of hospitalized patients with SARS-CoV-2 infection was 54 years, and 22.2% patients were aged > 65 years [11] . Patients with persistent SARS-CoV-2 RNA positivity were older. Although age >65 years was considered as a risk factor associated with disease severity [2] . However, our study did not find age to be a risk factor for viral clearance. The key difference in age between the two groups may be related to weak immune response as well as coexisting illnesses.

A majority of COVID-19 patients had hypertension and diabetes, and a small proportion had chronic lung disease, cardiac disease, immunosuppression or pregnancy. COVID-19 patients with underlying comorbidities, including hypertension, diabetes, cardiovascular disease, were more likely to be transferred to ICU [12] . Underlying diseases has been shown to be related to prolonged viral shedding in SARS patients [6] .

However, we found no significant differences between virus-positive patients and virusnegative patients with regard to comorbidity. This result suggests that underlying diseases such as hypertension and diabetes may be related to the patient's disease prognosis but have little to do with the duration of viral shedding.

Male sex was found to be an independent risk factor for prolonged SARS-CoV-2 viral shedding. In SARS-CoV infection, males experience higher mortality than females [13] .

A c c e p t e d M a n u s c r i p t Imbalanced levels of angiotensin-converting enzyme 2 (ACE2) between males and females may play an important role in the sex-based differences in the response to the disease [14] . ACE2 has been identified as the host receptor of SARS-CoV, which regulates both cross-species and human-to-human transmissions in SARS-CoV infection [15] . SARS-CoV-2 also use the ACE2 receptor to facilitate viral entry into target cells [16] .

Consistent with other reports, the most common laboratory abnormalities among the COVID-19 patients were depressed total lymphocyte and lymphocyte subset counts, as well as an elevated CRP level [2, 3, 17] . Compared with the virus-negative patients, patients with persistent SARS-CoV-2 positivity had lower lymphocyte count and T-cell subset count but higher CRP and PCT level. This suggested more severe cellular immune deficiency and inflammatory activation in SARS-CoV-2 virus-positive patients.

Decreases in T cell counts were strongly associated with the severity and progression of SARS and MERS in patients [18, 19] . The underlying reasons for lymphopenia may be the direct infection of lymphocytes by SARS-CoV-2, lymphocyte sequestration in the lung or cytokine-mediated lymphocyte trafficking [20, 21] . All of which may lead to extend the virus clearance time.

Although various drugs have been tested for COVID-19, there is still an urgent need for a clinically proven, effective antiviral treatment for SARS-CoV-2 infection. In this study, the antiviral therapy mainly included LPV/RTV and arbidol. Nevertheless, we A c c e p t e d M a n u s c r i p t observed that there was no apparent effect on viral shedding from the use of theses antiviral drugs or their combined use. Previous researches on the efficacy of LPV/RTV in SARS and MERS showed disparate results. Compared with those using ribavirin alone, improved outcomes for SARS and decreased viral loads were reported for patients using a combination of LPV/RTV and ribavirin [22] , whereas no obvious antiviral effect against MERS-CoV with lopinavir was observed in vitro [23] . Another study revealed that LPV/RTV could improve pulmonary function without inhibiting virus replication in MERS-CoV infection [24] . Current evidence regarding the effect of arbidol against SARS-CoV-2 was rare. In vitro experiment have indicated that arbidol showsed some antiviral activity against SARS [25] . It was previously demonstrated that remdesivir had excellent antiviral activity against MERS-CoV in vitro and in vivo [24] .

Moreover, remdesivir exhibited a beneficial effect in the first case in America [26] . This novel nucleotide analogue had not been put into use in our center yet, while its therapeutic effect in illness improvement is worth attention. Overall, further studies are warranted to uncover the exact efficacy of these different antiviral agents on viral shedding.

Corticosteroids have been used frequently for the treatment of severe patients infected with coronavirus by reducing inflammatory-induced lung injury [3] . Several studies have reported that the use of corticosteroids is associated with delayed viral RNA clearance in MERS or SARS patients and even with higher mortality in influenza pneumonia [27] [28] [29] . In this study, corticosteroid usage did not independently increase A c c e p t e d M a n u s c r i p t the risks for viral shedding regardless of the initial dosage or an earlier implementation.

However, the percentage of corticosteroid use in our center reached up to 77%, and the dosages prescribed were relatively lower for these COVID-19 patients than for MERS and SARS patients in previous studies. It was reported that high dosages of corticosteroids (>150 mg/d) showed a similar influence on H7N9 infection with prolonged viral shedding time, whereas proper use of corticosteroids was associated with clinical improvement in SARS [30, 31] . Thus, more clinical data are urgently needed to indicate the effects of corticosteroids and the optimal scheme of their usage.

In addition, the beneficial effect of immunoglobulin has also been observed in viral infection [32] . Our study found immunoglobulin application was associated with delayed viral clearance. In fact, patients using immunoglobulin were more likely to be in the more severe condition. Thus, more studies should be done to confirm the association between immunoglobulin treatment and viral shedding.

We identified the predictive value of the APACHE II score in SARS-CoV-2 infection.

Moreover, patients transferred to the ICU showed delayed SARS-CoV-2 RNA clearance. The result indicated that SARS-CoV-2 viral shedding was associated with disease severity. Similarly, MERS-CoV sub-genomic mRNA was detected more frequently in the severe group than in the mild group [5] . Severe patients had more prolonged MERS-CoV shedding, up to 18-27 days following the onset of symptoms [33] . SARS patients with APACHE II scores ≥ 20 were associated with an increased risk of transmission of SARS-CoV [34] . It can be explained by the higher A c c e p t e d M a n u s c r i p t APACHE II score, more severe immunosuppression and higher viral load among patients in the ICU.

Fecal-oral transmission was considered as an additional route for SARS-CoV-2 spread.

In this study, we found a high percentage of COVID-19 patients with positive fecal virus results, who needed more time to clear SARS-CoV-2 than those with negative fecal virus results. Fecal viral RNA can remain positive even after respiratory specimens test results return negative. Consistent with our results, stool specimens tested positive in up to 53% of COVID-19 patients, and the positive staining of ACE2 and SARS-CoV-2 nucleocapsid protein was observed in gastrointestinal epithelium as well as isolated infectious SARS-CoV-2 from feces [4] . The duration of SARS fecal viral excretion could last for more than 100 days [6] . Positive fecal viral RNA was highly suggestive of virus intestinal infection; thus, a longer isolated observation time should be considered in these COVID-19 patients.

Our study has some notable limitations. First, we only observed the hospitalization of COVID-19 patients for 28 days, and many patients remained in hospital with SARS-CoV-2 RNA positivity. The duration of viral shedding may be longer than this cut-off time point. Second, some patients were transferred to our hospital after the disease had progressed and become worse; hence, our cohort might represent a more severe range of COVID-19.

A c c e p t e d M a n u s c r i p t

In conclusion, we found that male sex, immunoglobulin use, APACHE II score, and lymphopenia were independent risk factors associated with the duration of SARS-CoV-2 RNA shedding, whereas ARV combination therapy and corticosteroid treatment were not. A prolonged SARS-CoV-2 RNA clearance time was associated with disease severity and fecal viral RNA positivity. Our findings suggest that more suitable antiviral agents and low-to-moderate corticosteroids rather than high dosages should be considered in the treatment of severe COVID-19, while seeking solutions to enhance immune function and to reduce the inflammatory response were also of great importance for viral clearance. In addition, more data based on randomized and controlled multicenter studies are needed to rigorously assess the clinical relevance of our proposed indicators. M a n u s c r i p t among patients who were transferred to ICU and those who were in general ward by day after illness onset. B, Cumulative proportion of patients with detectable SARS-CoV-2 RNA among those whose APACHE II score >10 and those whose APACHE II≤10 by day after illness onset. C, Cumulative proportion of patients with detectable SARS-CoV-2 RNA among those whose lymphocyte count ≤0.5ⅹ10 9 /L and those whose lymphocyte count > 0. A c c e p t e d M a n u s c r i p t M a n u s c r i p t 

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This study was funded by Science and Technology Department of Zhejiang Province 

A c c e p t e d M a n u s c r i p t