Symptomatic Infection is Associated with Prolonged Duration of Viral Shedding in Mild Coronavirus Disease 2019: A Retrospective Study of 110 Children in Wuhan

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subsets (T cells, B cells and natural killer cells), cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, tumor necrosis factor [TNF]-α and interferon [IFN]-γ) and inflammatory factors (hypersensitive C-reactive protein [hs-CRP]
, ferritin and procalcitonin) on admission. Chest radiologic findings were obtained on admission or the most obvious findings during hospitalization. The dates of illness onset, last exposure, diagnosis and discharge of patients were also recorded. Inpatients were discharge when they met the appropriate criteria, 2 one of which was testing negative for SARS-CoV-2 RNA by a throat or nasopharyngeal swab specimens two continuous times (with an interval time of more than 24 hours). Thus, we regarded the time from illness onset to discharge as the duration of viral shedding of SARS-CoV-2 in symptomatic children. Asymptomatic children came to the hospital due to an exposure history or abnormal chest radiologic imaging. Therefore, we regarded the time from dates of last exposure or abnormal chest radiologic imaging to discharge as the duration of viral shedding in asymptomatic children.

Statistical Analysis
Categoric variables were displayed as numbers and percentages. Continuous variables were presented as median (interquartile range [IQR]) and were compared using independent group t-tests when the data were distributed normally. The Mann-Whitney test was used when the data were not normally distributed. Categoric variables were compared using the χ 2 test or Fisher's exact test when the data were limited. Univariate and multivariate analyses with stepwise logistic regression were performed to identify risk factors for symptomatic infection. The cutoff point was selected from the median or quartile of each candidate risk factor. Prolonged duration of viral shedding was analyzed by the Kaplan-Meier method and the stratified log-rank statistic in different groups. Symptoms, abnormal laboratory markers, and pneumonia were used as potential factors affecting the duration of viral shedding, and the negative conversion of SARS-CoV-2 was selected as the event endpoint. Statistical analyses were performed using SPSS Software version 23.0. Statistical significance was defined as a two-sided P-value of less than 0.05.

Demographic and Clinical Characteristics
A total of 110 children with mild or ordinary COVID-19 were included in the current study, including 81 (73.6%) symptomatic patients and 29 (26.4%) asymptomatic patients. The median age of these patients was 6 years (range, 2 months to 15 years). An approximately 1.2:1.0 male-to-female ratio was found. In symptomatic patients, the most common symptoms were cough and dyspnea (51.8%), followed by fever (50.9%). Notably, 26 (23.6%) patients had digestive symptoms. All patients were administered antiviral therapy, of which interferon-α nebulization was the most frequently used. None of the patients required oxygen therapy. Detailed information is shown in Table 1.
We compared laboratory results between symptomatic patients and asymptomatic patients and found no difference except for slight anemia (P = 0.022), and slightly increased levels of aspartate aminotransferase (P = 0.004), which were observed only in symptomatic patients (Table 2).

Risk Factors Associated with Prolonged Viral Shedding
The median duration of viral RNA shedding was 15 days (IQR 11-20 days), ranging from 5 to 37 days. The duration of viral shedding in symptomatic patients (17 [IQR 12-23]) was longer than that in asymptomatic patients (11)(12)(13)). In Kaplan-Meier analysis, median age, symptoms, pneumonia, lymphocyte quartile and upper or lower range limits of laboratory markers were used as cutoff points for potential risk factors associated with prolonged duration of viral shedding. It was found that symptomatic infection (P < 0.001), fever (P = 0.006), pneumonia (P = 0.003) and lymphocyte counts <2.0 × 10⁹/L (P = 0.008) were associated with prolonged duration of viral shedding in children with COVID-19 ( Fig. 1).

Relationship Between Symptomatic Infection and Clinical Features
It was found that age younger than 6 years, lymphocyte counts <2.0 × 10⁹/L, globulin < 20.0 g/L, hs-CRP > 3.0 mg/L, procalcitonin > 0.05 ng/mL, IL-10 > 5.9 pg/mL, CD4+/CD8+ T cell ratio < 0.96 and pneumonia were associated with symptomatic infection in univariate analysis. In multivariate analysis, age younger than 6 years, hs-CRP beyond normal and pneumonia were independent risk factors for symptomatic infection in children with COVID-19 (Table 3). There were 64 (62.1%) patients who presented with pneumonia and these were more common in symptomatic patients (P < 0.001). The association between symptoms and pneumonia was evaluated using univariate analysis. It was found that symptomatic infection, fever, cough, dyspnea and digestive symptoms were correlated with pneumonia among children with COVID-19 (Table 4). Nevertheless, multivariate analysis showed no significant difference.

DISCUSSION
In the present study, the median duration of SARS-CoV-2 RNA shedding was 15 days. Prolonged viral shedding was associated with fever, pneumonia and lymphocyte counts < 2.0 × 10⁹ in children with a mild and ordinary type of COVID-19. In addition, symptomatic infection was associated with an increased risk of pneumonia. Furthermore, younger age (<6 years) was an independent risk factor for symptomatic infection in children with COVID-19.
In the current study, all the asymptomatic children at admission were mild or ordinary COVID-19, which is consistent with asymptomatic adults. 9 Upper respiratory specimens have shown higher viral loads in symptomatic adult patients soon after symptom onset, which gradually decreased. 6 In the present study, the duration of viral shedding in symptomatic children was significantly longer than in asymptomatic cases, which indicated that symptomatic patients had much more transmission potential and may need to be isolated.
In reports of adult inpatients with COVID-19, most of them were symptomatic and presented with radiologic evidence of pneumonia. 10,11 Invasive mechanical ventilation was associated with prolonged viral shedding in adult patients with SARS-CoV-2 infection. 12 In addition, higher viral RNA load and longer viral shedding of influenza A can be detected in lower respiratory tract specimens compared with upper respiratory tract specimens. 13 Therefore, patients with pneumonia might have a high viral load in lower respiratory tract specimens, and have longer for viral clearance times.
In this study, symptomatic infection was more common in younger children. For young children who had an exposure history and presented fever, it is important to perform throat or  (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) 17 (12-23) 11 (9-13) <0.001 NA, not available; IQR, interquartile range. Symptoms of digestive system included diarrhea, vomiting, poor feeding/anorexia and abdominal pain. Symptoms of central nervous system included headache. Symptoms of muscular system included malaise. nasopharyngeal swabs to confirm SARS-CoV-2 infection. It is worth noting that digestive symptoms such as diarrhea, vomiting, poor feeding and abdominal pain were not rare in children with COVID-19, which should not be ignored by parents and doctors.
Children with COVID-19 experienced a milder clinical course than adults, and the majority of children recovered with interferon-α nebulization, similar to children with SARS. 14,15 There are some limitations to this study. First, this was a retrospective study, and not all tests were performed and monitored during hospitalization in all patients. Second, all patients were diagnosed by throat or nasopharyngeal specimens using RT-PCR, but other specimens such as feces and urine were not monitored for SARS-CoV-2. Lastly, the ability to associate symptoms with duration of viral shedding might be limited by the sample size.

CONCLUSIONS
We identified that prolonged duration of viral shedding was associated with fever, pneumonia and lymphocyte counts less than 2.0 × 10⁹/L in children with mild and ordinary COVID-19.