Severe acute respiratory syndrome coronary virus-2 (SARS-CoV-2) has rapidly ranged, domestically, to all of China, and globally, to many countries across 6 continents. WHO named the illness associated with SARS-CoV-2 as the 2019 coronavirus disease (COVID-19). SARS-CoV-2 is a large family of viruses that cause illness ranging from the mild upper respiratory tract to more severe diseases, such as MERS and SARS.1 The spread of COVID-19, similar to MERS and SARS, between people has generally occurred between close contacts. According to the WHO, common signs of COVID-19 infection in adult include respiratory symptoms, fever, cough, shortness of breath and breathing difficulties. Serious cases can lead to pneumonia, severe acute respiratory syndrome, kidney failure and death.2 To contrast with infected adults, most infected children appear to have a milder clinical course, while only 1 patient died.3 Disease severity is a particularly crucial parameter for understanding this new disease, but accurately estimating the case-fatality risk is difficult because milder cases are not being diagnosed and death is delayed.4
Despite the worldwide spread, little is known about the severity or death in children. In this study, we explored the risk factors of in-hospital severity for patients and describe the clinical course of symptoms, viral shedding and temporal changes of laboratory findings during hospitalization in pediatric patients with COVID-19 in a tertiary hospital in China.
Study Population and Design
A retrospective case-control study was taken in children with SARS-CoV-2 infection in Wuhan Children’s Hospital. We reviewed the medical records of hospitalized children in general ward and intensive care unit between January 25, 2020 and March 8, 2020. Cases were defined as children diagnosed with severe COVID-19 pneumonia according to Chinese Clinical Guidance. In detail, severe pneumonia met any of the following criteria: (1) increased respiratory rate: ≥70 times/min (<1 year), ≥50 times/min (≥1 year) (after ruling out the effects of fever and crying); (2) oxygen saturation <92%; (3) hypoxia: assisted breathing (moans, nasal flaring and 3 concave sign), cyanosis, intermittent apnea; (4) disturbance of consciousness: somnolence, coma, or convulsion; (5) food refusal or feeding difficulty, with signs of dehydration.5 Controls with mild respiratory symptoms (fever and/or cough), laboratory-confirmed COVID-19 infection, matched for age (±6months), sex and date of admission (±3 days), and who classified as non-severe type, were selected from the hospital admissions. All the included children were diagnosed COVID-19 with a confirmed SARS-CoV-2 RNA detection in nasopharyngeal swabs.
This study was approved by the Ethics Committee of Wuhan Children’s Hospital of Tongji Medical College of Huazhong University of Science and Technology and written informed consent was waived due to the rapid emergence of this infectious disease.
Laboratory and Radiologic Procedures
Nasopharyngeal swabs were taken for detection of SARS-CoV-2 RNA on admission and throughout the clinical course. Laboratory detection of SARS-CoV-2 RNA was detected by reverse real-time polymerase chain reaction as previously described.6 The criteria for discharge were absence of fever for at least 3 days, symptoms of dyspnea and cough significantly improved, and nasopharyngeal swab was negative for detection of SARS-CoV-2 RNA 2 consecutive times (1-day sampling time interval at least).
There are other laboratory tests performed on admission to evaluate the status of COVID-19 infection, including blood routine counts, blood gas analysis, liver and kidney function, inflammatory biomarkers [IL-6, IL-10, high sensitive C reaction protein (hsCRP)], biologic enzymes (lactate dehydrogenase, creatine kinase and creatine kinase-MB), D-dimer, coagulation image and urine routine test.
To investigate the immune response against COVID-19, we looked at peripheral blood mononuclear cells (PBMCs) including T lymphocyte cells, B lymphocyte cells and natural killer (NK) cells. On admission, PBMCs are obtained from ethylenediaminetetraacetic acid anticoagulant whole blood via erythrocyte lysis. Then, flow cytometric analysis was used to determine percentages of T cells and their subsets (CD3+, CD4+, CD8+, CD25+), B cells (CD19+) and NK cells (CD16+/56+) in PBMCs. Chest CT and radiograph images from cases on the first day of admission seen at Wuhan Children’s Hospital were analyzed by 2 radiologists independently. The evaluators assessed the CT features using both axial CT images and multiplanar reconstruction images. The distribution of the lesion was recorded according to bronchopulmonary segments.
The medical records of included children were retrieved retrospectively. Epidemiologic, symptoms, clinical features, laboratory test, chest CT scan imaging data, treatment therapy and respiratory support were obtained for subsequent analysis. The hospital stay, ICU admission were also recorded. The data were reviewed by 2 independent physicians (C.W. and F.Z.).
Normally distributed continuous variables were presented as mean (SD), non-normally distributed variables were described as median and interquartile range (IQR) and the Mann-Whitney U test was used. Categorical variables were presented as frequency rates and percentages. Categorical variables were compared using the χ2 test or Fisher exact test. We use SPSS version 22.0 software for all statistical analyses. A 2-sides P-value of less than 0.05 was considered statistically significant.
Demographic and Clinical Characteristics
By March 14, 2020, 260 children who were admitted to Wuhan Children’s Hospital which was a designated center for quarantine and treatment of COVID-19, of whom 8 children diagnosed with severe COVID-19 pneumonia were included in the study. Thirty-five children with COVID-19 infection matched for age, sex and date of admission, and who classified as non-severe type. The median age of severe cases was 5.06 years (IQR 0.97–13.83), and 6 (75.0%) of them were boys (Table 1). Among 8 severe cases, 2 (25.0%) children were found to have a comorbidity. One child with severe COVID-19 pneumonia, 8-year-old boy, had been diagnosed with acute lymphoblastic leukemia in remission before. The other one in severe status, 15-year-old boy, has been diagnosed primary obesity with body mass index of 33.1. However, the comorbidity status was not a risk to develop the poorer outcome. Because the comorbidities were found in 8 (22.9%) of the non-severe group and the probability of coexisting underlying diseases were not different between severe group and non-severe group (P = 1.00).
For cases with severe pneumonia caused by COVID-19, all of them had symptoms of respiratory tract. The most common symptoms were dyspnea (87.5%), fever (62.5%) and cough (62.5%). And 3 cases (37.5%) developed symptoms of the digestive tract including vomiting and diarrhea. In non-severe group, 31 (88.6%) developed fever, 31(88.6%) had cough and 8(22.9%) had diarrhea, after exposure to her/his infected family members.
Laboratory and Radiologic Findings
We tracked major laboratory markers since illness onset. On admission, white blood cells count was significantly higher in severe children than non-severe children. Levels of inflammation bio-makers such as hsCRP, IL-6, IL-10 and D-dimer elevated in severe children compared with non-severe children on admission (Table 1). Biomarkers of liver function showed normal and similar in both groups, except total bilirubin (TBIL) elevated in severe children. Among the biomarkers of kidney function, only the level of uric acid clearly elevated in severe children compared with non-severe children on admission. Meanwhile, no difference of biologic enzymes was observed between severe and non-severe groups.
Although lymphocyte count was similar severe children and non-severe children in routine blood count, the counts of the subsets of lymphocyte NK (CD16+/56+) cells in severe children were significantly higher than those in non-severe children according to flow cytometric analysis (Table 1). We did not observe the differences of the counts of the other subsets of lymphocyte between severe and non-severe groups.
As expected, all of severe children displayed the lesions on chest CT or radiograph on admission, on contract more than half of non-severe children (51.4%) had abnormal findings in radiologic examination. More lung segments were involved in severe children than in non-severe children. The media number of involved lung segments of severe children was 5.50 (IQR 4.30–11.80), and the number was 1.50 (IQR 1.00–4.30) in non-severe children (P = 0.003). Although all lung segments can be involved, there was a strong predilection for the left lower lobe in both severe and non-severe children (lesions in right lower lobe found in 75.0% severe children and 27.6% non-severe children). Among severe children, the predominant manifestations of chest CT were ground-glass opacity, which was found in 3 (37.5%), patchy consolidation in 2 (25.0%). For non-severe children, the ground-glass opacity was most common findings (45.7%) on chest CT on admission, and patchy consolidation was less frequent involved compared with severe children (20.0%).
Treatment and Outcome
All severe children received oxygen therapy, 5 (13.5%) of them were put on noninvasive mechanical ventilation, 3 (8.1%) of them had endotracheal intubation and put on invasive ventilation under progressive hypoxia. In comparison, none of the children in non-severe group was received oxygen therapy. The median duration from onset of symptoms of severe cases was 3.00 (1.00–6.75) days. The median time for nucleic acid tests turning negative was 10.50 (6.75–13.75) in severe children, which was significantly longer than that in non-severe children [7.10 days (IQR 4.00–11.50)]. Most of severe children recovered with 1 patient died. Average hospital stay was 13.50 days (IQR 12.00–17.30) for severe children, which was longer than average hospital stay of 11.00 days (IQR 10.00–16.00) for non-severe children.
Risk Factors for Developing Severe Pneumonia
The association of clinical factors with severe pneumonia is summarized in Tables 1 and 2 by univariate and multivariate logistic regression. In univariable analysis, Uni versus bilateral pneumonia, elevation of NK cell and increasing of TBLL associated with the severe COVID-19. In multiple univariable analysis, more than 3 lung segments involved was only risk factor associated with severe COVID-19 pneumonia (odds ratio = 25.0, 95% confidence interval 2.51–248.57, P = 0.006).
This retrospective cohort study revealed that most of the children patients manifested mild symptoms. We identified several risk factors for severity in children who were hospitalized with COVID-19. In particular, the activation of immune cells including of elevated white blood cell, hsCRP, and activated IL-6, IL-10, NK and the higher INR, D-dimer. Moreover, abnormal TBLL, uric acid (UA) and D-dimer also involved in the aggravation in the clinic in pediatric COVID-19.
The risk factors related to the development of COVID-19 of children and progression included organ and coagulation dysfunction (eg, higher TBIL, UA and INR, D-dimer). Similar to the adults with COVID-19, intravascular coagulation was on the pathway to severity in children patients.7 No clinic signs involved to be a potential risk factor of severe COVID-19 in children, while the value of TBIL and UA increasing might indicate the damage in the bile duct, liver and kidney.8 Otherwise, lung injury is beyond doubt in COVID-19 patients of both adult and children. CT scan is vital to evaluate the damage of lung. All of severe children patients manifested CT image positive at the first day of admission, increased number of damaged lung segments, damage to both lungs were associated with COVID-19 disease severity in children. Further study found the most vulnerable fragments in turn are left lower lung, right upper lung, right lower lung, rather than just lower lobe. This is deviant with the adult who were concentrated in lower lobe.9 The univariable analysis in our present suggested more than 3 lung segments was the high-risk factor associated with severe COVID-19 in children. Similar to the adult acute respiratory distress syndrome, all of the severe patients demonstrated obviously dyspnea, with CT over 3 segments positive. Interestingly, the recovery of hypoxia is also accompanied by improvement of CT lesions including reduction in lesion size and severity. This reveals that CT represents pathologic changes, associated with the symptom and severe in pediatric COVID-19.
All pediatric patients had a history of exposure to a familial member with confirmed SARS-CoV-2 infection. However, age, sex and clinic signs did not involve to be a potential risk factor of severe COVID-19 in children. In immune changes, the activated T cell-induced the disease severe. Higher lever CRP and increased white blood cell indicated the co-infection in children COVID-19 is severe. Otherwise, it is completely opposite to the adult patients with the lymphopenia and neutropenia in more than half of the SARS patients at the initial phase of their infection.10 Furthermore, the NK cell and the cytokine produce by T lymphocyte, such as IL-6, IL-10 apparently activated in severe patients, while lower expression in common children patients of COVID-19. The elevation of IL-6 was protective to the host defending against bacterial by down-regulating the activation of the cytokine network.11,12 The univariable analysis in our present found the elevation of IL-6 was associated with the severe children patients, which indicated the activated immune cell and cytokine protected to the host defending against SARS-Cov-2 in severe condition.
None of the children in non-severe group received oxygen therapy. Although all of the severe children patients of COVID-19 received oxygen therapy, 3 patients put on invasive ventilation under progressive hypoxia. Surprisingly, only 1 patient died, who was a 8-year-old boy, accompanied with acute lymphoblastic leukemia in remission. Similar to adult, nucleic acid tests turning negative in severe children was significantly longer than that in non-severe children. Which indicated the longer viral loading in patients, the more serious COVID-19.
Limitation is small number of severe COVID-19 in children. However, children patients characterized by mild clinic signs and rare mortality.13 There were only 8 severe patients in hospitalization of 260 COVID-19 of children. Lower proportion of severe patients might partly induce bias. However, these are already very comprehensive data as a only designated hospital for children COVID-19.
From the discussion above, we can identify some factors that may associate to the severe in SARS-CoV-2 infected children. Most importantly, CT lesion over 3 segments indicate the severity of lung damage in pathology, which is the highest risk to the aggravation of COVID-19 in children. Second, the over-activated immune response to SARS-CoV-2, especially IL-6 elevation associated to the severity. Finally, the organ damage, such as hepatobiliary and intravascular coagulation dysfunction might suggest multiple organ dysfunction.
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