Clinical Characteristics of Mild/moderate COVID-19 Patients with a Prolonged Negative Conversion Time of SARS-CoV-2 Nucleic Acid Detection

Ya Yang The First A liated Hospital of Army Medical University Xiaogang Hu Chongqing university cancer hospital Lirong Xiong The First A liated Hospital of Army Medical University Peishu Fu The First A liated Hospital of Army Medical University Wei Feng The First A liated Hospital of Army Medical University Wei Li The First A liated Hospital of Army Medical University Liwen Zhang Chongqing Public Health Medical Center Fengjun Sun (  fengj_sun@163.com ) The First A liated Hospital of Army Medical University

lungs in the later stage. COVID-19 patients also could be asymptomatic with no associated clinical manifestations, but the SARS-CoV-2 RNA test was positive.
According to the severity of the disease, COVID-19 patients were were divided into asymptomatic, mild/moderate and severe cases. Without timely treatment, mild/moderate patients will develop into severe cases, increasing the fatality rate. It is very important to control the disease progression of mild/moderate patients. The standard of cure is the relief of symptoms and two successive negative viral nucleic acid in the "Diagnosis and Treatment Scheme of New Coronavirus Infected Pneumonia" (trial version 7) [6]. The negative conversion of SARS-CoV-2 RNA was essential in the discharge criteria during hospitalization [7,8]. The negative conversion time (NCT) of SARS-CoV-2 RNA is closely related to clinical manifestation and disease progression in COVID-19 patients. Hu et al. reported that NCT of SARS-CoV-2 RNA occurred in 14 days (IQR: [10][11][12][13][14][15][16][17][18]. Older age and chest tightness were independently associated with delayed clearance of SARS-CoV-2 RNA in hospitalized patients [9]. The COVID-19 patients with digestive symptoms are more likely to test positive for viral RNA in a stool sample and have a longer delay before viral clearance than patients with only respiratory symptoms [10]. Preliminary clinical treatment showed that some drugs, such as chloroquine, effectively potentiated virus clearance in COVID-19 patients [11].
However, there is limited data regarding the potential predictors of NCT in mild/moderate COVID-19 patients.
Therefore, it was necessary to identify systematically factors associated with prolonged NCT in mild/moderate COVID-19 patients. This study analyzed clinical characteristics of mild/moderate COVID-19 patients with prolonged NCT of SARS-CoV-2 RNA, which provide a useful references for disease progression and treatment of COVID-19.

Subjects and data collection
The clinical data and laboratory test results were analyzed retrospectively from 32 mild/moderate COVID-19 patients admitted to the Public Health Medical Center and Prevention of Chongqing from January 31 to February 28, 2020. All patients were identi ed to be nucleic acid positive for SARS-CoV-2 and were convalescent before hospital discharge.
Data was collected and recorded from each patient, which included demographic and clinical information (gender, age, smoking history, and comorbidities), clinical manifestations (fever, coughing, expectoration, anhelation, myalgia, nausea and vomiting, diarrhea, osphyalgia, etc.), and laboratory test results (standard blood counts, procalcitonin, C-reactive protein, blood biochemistry, coagulation function, and myocardial enzyme spectrum, etc). The normal range of laboratory examination is the standard of Reference Range Values for National Practice for Clinical Testing (4th Edition) published by People's Medical Publishing House [12]. Chest CT and therapeutic drug use were also documented. Then, the factors associated with NCT of SARS-CoV-2 RNA in upper respiratory specimens were analyzed.

Therapeutic strategies
Strategies of treatment for COVID-19 patients included antiviral treatment and symptomatic treatment.
The primary treatment strategy for COVID-19 is combined antiviral therapy, which mainly comprises interferon-α2b IFN-α2b and lopinavir / ritonavir, or IFN-α2b and ribavirin. When SARS-CoV-2 RNA was still detectable after 19 days of drug combination therapy, chloroquine phosphate was added to the antiviral treatment.
De nitions of basic concepts Sputum and throat swab specimens were collected from patients. RT-PCR assay was used to detect genes of SARS-CoV-2, which were performed twice in every 24 h period. Patients with consecutive positive nucleic acid tests were con rmed as SARS-CoV-2 infection. The standard of negative conversion is two successive negative viral nucleic acid detection tests in 24 h minimum sampling intervals. The term convalescent patients refer to recovered afebrile patients without respiratory symptoms who had two successive (minimum 24 h sampling interval) negative results for SARS-CoV-2 RNA from oropharyngeal swabs by RT-PCR.

Statistical analysis
Normally distributed continuous variables are summarized as the mean mean (standard deviation, SD), otherwise, median (interquartile range, IQR) was used to describe. Categorical variables are expressed using numbers and percentages. Cox regression analysis was used to analyze the factors. First, univariate analysis was performed, and the indicators with statistical signi cance were analyzed for Kaplan-Meier survival analysis. The Log Rank method was used to compare the differences between groups. A Cox proportional hazard model was used for multivariate analysis. IBM SPSS Statistics 26 was used for statistical analysis and survival image rendering. P<0.05 was considered to indicate a statistically signi cant difference.

Results
The relationship between demographic and clinical information and NCT of SARS-CoV-2 RNA Of these patients, males accounted for 43.8% (14) of con rmed cases, and 56.3% (18) were females. The median age of males and females was 34.00 (29.00, 47.00) years and 43.00 (37.75, 57.25) years respectively, suggesting that middle-aged people were more susceptible to infection than other age groups. A total of 12.5% of patients were smokers. More than a quarter of patients (34%) had underlying diseases, including cardiovascular and cerebrovascular diseases (25%), gastrointestinal disease (15.6%), respiratory disease (12.5%), endocrine disease (9.3%), a neurological disorder (3.1%) and urinary disease (3.1%). All patient data were analyzed using the Cox regression analysis for univariate analysis. However, we found that no demographic or clinical information had a signi cant positive association with NCT of viral RNA in patients (P > 0.05, Table 1). Data are shown as n (%) or IQR unless speci ed otherwise.
The Log Rank method was used to compare the differences between groups, p < 0.05 was considered to indicate a statistically signi cant difference (indicated by *).

The relationship between clinical manifestation and NCT of SARS-CoV-2 RNA
The median duration from disease onset to hospital admission was 3.5 (IQR: 2-6) days, with a median of 20 days from illness onset to hospital discharge (IQR: 15.25-26) and the median time from positive to negative conversion of SARS-CoV-2 RNA was 19.5 days (IQR: 14.25-25). The majority of the patients showed an initial symptom of fever (75%), but a quarter of the patients were afebrile, alerting the need for the caution of atypical cases. In addition to fever, other clinical symptoms mainly included fatigue (78.1%), expectoration (56.3%), diarrhea (68.8%), nausea (37.5%), anhelation (34.4%), and throat discomfort (34.4%). Clinical manifestations of the study population are summarized in Table 2. Data are shown as n (%) unless speci ed otherwise.
The Log Rank method was used to compare the differences between groups, p < 0.05 was considered to indicate a statistically signi cant difference (indicated by *).
The Cox regression analysis was used for univariate analysis; we found that fever, nausea, and diarrhea were a signi cant positive association with NCT of viral RNA in patients (P < 0.005, Table 2). The KaplanMeier curves revealed that fever, nausea, and diarrhea had a signi cantly prolonged NCT of SARS-CoV-2 RNA compared with the normality's group (P < 0.05; Fig. 1A, B, C).
The relationship between laboratory ndings and NCT of SARS-CoV-2 RNA As shown in Table 1, the laboratory inspection section showed that 43.8% of the patients developed leucopenia, and 28.1% of patients had lymphocytopenia. The hemoglobin, platelets, and albumin levels were lower in 53.1%, 37.5%, and 34.4% of patients, respectively. The levels of procalcitonin, C-reactive protein, total bilirubin, direct bilirubin, creatinine, lactic dehydrogenase were increased in 9.4%, 40.6%, 40.6%, 46.9%, 25%, and 21.9% of patienst, respectively. All patient data were analyzed using the Cox regression analysis for univariate analysis. Still, there were no signi cant ndings with a positive association with NCT of viral RNA in patients (P > 0.005, Table 3). Data are shown as n (%) unless speci ed otherwise.
The Log Rank method was used to compare the differences between groups, p < 0.05 was considered to indicate a statistically signi cant difference (indicated by *).
The relationship between radiological ndings and NCT of SARS-CoV-2 RNA Ground-glass opacity (GGO) and high-density shadow were the typical radiological ndings on chest CT scan. Abnormalities in chest CT were detected in 93.8% of patients. A total of 87.5% of patients had ground-grass opacities over bilateral lungs, which was the most common pattern of CT changes and corresponded to pathological diffuse alveolar damage [13].
We analyzed the univariate analysis using the Cox regression and found that abnormalities in chest CT were a signi cant positive association with NCT of viral RNA in patients (P < 0.005, Table 4). The Kaplan Meier curves revealed that abnormalities in chest CT had a signi cantly prolonged NCT of SARS-CoV-2 RNA compared with normality's group (P < 0.05; Fig. 1D). Table 4 The relationship between radiological ndings a and NCT of SARS-CoV-2 RNA in 32 patients with COVID-19 Data are shown as n (%) unless speci ed otherwise.
The Log Rank method was used to compare the differences between groups, p < 0.05 was considered to indicate a statistically signi cant difference (indicated by *).
a How many patients have the following lesion location.

Taking antiviral medications during hospitalization
All patients received antiviral therapy during hospitalization. All 32 patients were treated with IFN-α2b.

Predictors of NCT
RNA. More importantly, fever and nausea were the independent factors predicting NCT of mild/moderate COVID-19 patients.
Fever, a respiratory manifestation, is the most commonly reported symptom in patients infected with SARS-CoV-2 [14,15]. Accordingly, fever may be a clinical sign of poor prognosis in patients and response to the release of in ammatory mediators such as cytokines and chemokines [16] [17]. These in ammatory mediators cause tissue damage and organ dysfunction by stimulating toxic oxygen derivatives suggesting that the NCT of SARS-CoV-2 RNA may be prolonged in patients [18,19].
Chest CT has a high sensitivity to detect lung abnormalities, which is quite helpful in the early diagnosis of the disease. The "Diagnosis and Treatment Scheme for Coronavirus Disease (Trial Version 7)" recommended that a CT examination serves as the diagnostic basis for COVID-19. For chest imaging in patients with COVID-19, the early manifestation was multiple plaque shadows and interstitial changes. The later development was multiple ground glass shadows and in ltration shadows in both lungs. In severe cases, lung consolidation can occur, presenting as "white lung," which may be related to the immunopathology. Most studies have suggested that a dysregulated/exuberant innate responses are the primary cause of coronavirus-mediated pathology [20]. Many cytokines or chemokines are involved in the immune storm after the infection of coronavirus,which eventually leads to lung injury and acute respiratory distress syndrome [21]. The improvement of chest CT was after that of body temperature. Still, it preceded the negative conversion of nucleic acid tests, suggesting that abnormalities in chest CT could indirectly re ect the persistence of SARS-CoV-2 RNA in patients with COVID-19. Our study found that abnormalities in chest CT could prolong the NCT of patients with COVID-19. Therefore, the improvement of chest CT as soon as possible has an essential effect on shortening NCT in patients with COVID-19 and promoting patients to be discharged more rapidly.
Although COVID-19 most commonly presents with respiratory symptoms, such as cough and shortness of breath [14,22,23], there is evidence that the illness can also present with nonrespiratory symptoms, most notably digestive symptoms such as diarrhea, diminished appetite, nausea, and vomiting [24][25][26]. Diarrhea appeared to be the most common GI complaint [27], followed by nausea and vomiting [22].
SARS-CoV-2 RNA was detected in stool samples from COVID-19 patients for the rst time, rst reported in the United States [28]. Viral RNA was still positive in gastrointestinal specimens even after levels could not be detected in respiratory samples [27], implying direct infectivity of the virus on the intestinal tract.
Current research shows that the primary target organ of COVID-19 is the lung, but clinical evidence suggests that the gastrointestinal tract may be another viral target organ.
The SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) has been found in both upper and lower gastrointestinal tract where its expression level was nearly 100 times higher than that of respiratory organs [29,30]. Patients with symptoms of the digestive system have more viruses in their gut [10], and maybe more likely to cause direct damage to the intestinal mucosa. Our research found that digestive symptoms, including nausea and diarrhea, are the factors of NCT of COVID-19 patients, suggesting that patients with gastrointestinal symptoms should seek medical care to avoid delayed diagnosis and prolong treatment time.
Chinese experts recommend that patients diagnosed as mild, moderate, and severe cases of COVID-19 pneumonia and without contraindications to chloroquine, be treated with 500 mg of chloroquine twice a day for ten days [31]. In our study, when SARS-CoV-2 RNA was still detectable after 19 days of drug combination therapy, chloroquine was added to the antiviral treatment. Thirteen patients were treated with chloroquine and a median duration of viral shedding of 6.0 (IQR: 5.0-7.0) days. Therefore, these results suggest that chloroquine may play a role in reducing viral load and shortening the time of virus negative transition. Other studies have also con rmed the role of chloroquine in promoting virus negative conversion and shortening the disease course. Other studies have also con rmed the role of chloroquine in promoting virus negative conversion, shortening the course of the disease [11] and reducing/eliminating viral load in COVID-19 patients [32]. However, we did not conduct a randomized controlled study to evaluate the e cacy of chloroquine and most infections with COVID-19 were selflimited, about 15% of infected adults developed severe pneumonia that required treatment [22,33], the numerical reduction in time to clinical improvement in those treated with chloroquine requires con rmation in more extensive studies.
There are several limitations to our study. First, the sample size of this study is not large enough. Second, we have not conducted randomized controlled trials, so we cannot determine the therapeutic effect of chloroquine on COVID-19 and its impact on shortening the time of negative viral conversion.

Conclusion
In conclusion, our study suggests that fever, nausea, diarrhea and abnormalities in chest CT hve a signi cantly prolonged NCT of SARS-CoV-2 RNA in mild/moderate COVID-19 patients. More importantly,

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.  Using of antiviral medications during hospitalization in 32 patients with COVID-19