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Gastrointestinal and Liver Manifestations in Children with COVID-19 and Their Relationship to Clinical Course

Objective: Coronavirus disease 2019 is a major health problem in all age groups. Although most clinical symptoms are respiratory, gastrointestinal symptoms are often reported. This is a major concern for children and has limited research coverage. In this study, we evaluated the frequencies of gastrointestinal symptoms and liver biochemical findings in children with coronavirus disease 2019 and their relationship with clinical course and length of hospital stay. Materials and Methods: Demographic data, clinical, and laboratory findings of children with Coronavirus disease 2019 who were followed up by the Department of Pediatric Infectious Diseases between March 2020 and August 2020 were recorded. They were classified according to age groups as <5 years, 5-10 years, and >10 years. Laboratory findings were analyzed according to age groups. Demographic, clinical, and laboratory findings were compared in both situations, the presence of gastrointestinal symptoms and the presence of elevated liver enzymes. It was considered statistically significant if it was <.05. Results: A total of 294 patients (median age 10 years [14 days to 18 years]) were enrolled in this study. Although fever is the most common symptom of coronavirus disease 2019, 15.6% of patients presented with acute gastroenteritis. Most patients with liver involvement (n = 130, 44.2%) were under 5 years of age (n = 74, 56.9%, P < .001). The patterns of abnormal liver test results were cholestatic (71.5%), hepatocellular (18.4%), and mixed (10%) types. Severe or massive elevation of aminotransferase or liver failure was not observed. No statistically significant difference was noted in outcomes, including length of stay, for patients with gastrointestinal symptoms (P = .178) or liver involvement (P = .146). Conclusion: The presence of gastrointestinal symptoms or elevated liver enzymes does not affect the course of the disease in children with coronavirus disease 2019.


INTRODUCTION
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is the global health threat of the century. Although COVID-19 affects all age groups, it often causes mild upper respiratory tract symptoms in children. 1 Severe acute respiratory syndrome coronavirus 2 requires the angio tensi n-con verti ng enzyme 2 (ACE-2) receptor to bind to the host cell. Immature immune systems and variations in the tissue expression of ACE-2 may lead to the development of different clinical signs in children. 2,3 The lung is the primary organ of involvement in SARS-CoV-2 infection, but extrapulmonary manifestations, such as gastrointestinal (GI) symptoms, have also been reported A mild rise in liver enzymes is commonly reported with COVID- 19. Although high levels of transaminases are considered to be associated with the severity of the disease, the pathological mechanism of liver injury has not been clarified. 7 Immunemediated mechanisms, endothelial cell infection and endotheliitis, vascular thrombosis, and direct cytopathic effects are considered possible causes in the pathogenesis of liver damage. 8,9 Because COVID-19 can decompensate underlying liver disease, patients with elevated transaminases should also be investigated for other liver diseases. 7,10 Rare manifestations of COVID-19 such as typical acute appendicitis, ileocolitis, and intussusception have also been reported as part of the clinical spectrum of SARS-CoV-2 disease in childhood. 11 In this study, it was investigated whether the presence of GI symptoms and the presence of elevated liver enzymes in patients affected the clinical course and hospital stay of COVID-19.

Study Design and Setting
This is a single-center, observational, retrospective casecontrol study conducted in the Pediatric COVID-19 Unit of the Faculty of Medicine at the University of Konya, Turkey, after receiving approval from the Selcuk University Faculty of Medicine Local Ethics Committee. Informed consent was obtained from the patient. Permission was also obtained from the Turkish Medicines and Medical Devices Institution, Republic of Turkey Ministry of Health (2020 -08-2 9T21-00-02 ).

Patients and Data
Using clinical data from March 22, 2020, to August 31, 2020, we retrospectively evaluated the data of 300 children who had been diagnosed with COVID-19 via real-time reverse trans cript ion-p olyme rase chain reaction (RT-PCR) testing. Six patients with GI comorbidities (e.g., celiac disease, cystic fibrosis, inflammatory bowel disease, autoimmune hepatitis, Wilson's disease, and metabolic liver disease) were excluded from the study. Thirteen patients had comorbidities (e.g., asthma, autoimmune disease, malignancy, immunodeficiency, or Canavan diseases). The others were previously healthy according to their family history. Children were divided into 2 subgroups according to the presence or absence of GI symptoms. They were then examined in 3 age groups (<5 years, 5-10 years, and >10 years). The following 5 clinical patient categories were applied according to symptoms and SARS CoV-2 RT-PCR positivity: 1. If the patient had SARS CoV-2 PCR positivity but no symptoms, it was considered an asymptomatic case. 2. If the patient had nasal stuffiness with discharge, sore throat, and cough, it was considered to be an upper respiratory tract infection (URTI). 3. If the patient had symptoms of cough, mucus, wheezing, and shortness of breath, but no pneumonia was found in the radiological examination, it was considered to be acute bronchitis. 4. If the patient had respiratory symptoms and radiological consolidation findings, it was considered to be pneumonia. 5. Acute gastroenteritis was applied if there was a rapid onset of diarrhea alongside other symptoms (e.g., nausea, vomiting, fever, and abdominal pain).
Laboratory parameters were evaluated at the patient's first admission to the hospital and prior to receiving medical treatment.
Serum AST, ALT, GGT, ALP, and bilirubin levels were evaluated according to normal reference ranges of the laboratory test kits recommended for our hospital. Serum liver tests were considered abnormal if levels were above the following thresholds: ALT > 35 U/L, AST > 35 U/L, GGT > 24 U/L, and total bilirubin (TBIL) >1.4 mg/dL. Serum ALP level was considered abnormal if it was >500 U/L in the 1-12 age group, >750 U/L in the 12-15 age group, or >150 U/L in those older than 15 years of age. Serum ALP and GGT values were evaluated for cholestatic liver injury if the ALP >1.5 × the upper limit of normal (ULN) and/ or GGT >3 × ULN, according to the European Association for the Study of the Liver guidelines. 12 Liver enzymes (e.g., AST and ALT), GGT, ALP, and TBIL were grouped by the upper limit of the normal range (<ULN) and abnormal range (>ULN). Aminotransferase elevations were defined as borderline (<2 × ULN), mild (2-5 × ULN), moderate AST/ALT 5-15 × ULN, severe AST/ALT >15 × ULN, and massive AST/ALT >10 000 IU/L. The R-factor value 13 was used to determine the likely type of liver injury (e.g., hepatocellular, cholestatic, or mixed) in patients with elevated aminotransferases and ALP or GGT. The R-factor value was calculated according to the formula, R = (ALT value/ALT ULN)/(ALP value/ALP ULN). In clinical situations where it is suspected that ALP elevations are from an extrahepatic source, GGT level is used. If the R-factor values are >5, 2-5, or <2, they are defined as hepatocellular, mixed, or cholestatic, respectively. 14,15 Serum samples from children with high liver enzymes were screened for hepatitis A, B, and C. Laboratory data on admission were also evaluated, including complete blood count with differential routine biochemical tests, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), D-dimer, troponin, international normalized ratio (INR), activated partial thromboplastin time (aPTT), and lactate dehydrogenase.
Children in all groups were evaluated in terms of demographics, admission symptoms, laboratory findings, and clinical outcomes.

Treatment Approach
All patients were isolated and given bed rest, and they were monitored for heart rate, blood pressure, and oxygen saturation. They also received supportive care. Hypoxemia and electrolyte imbalances were treated with supplemental oxygen and fluid replacement therapy according to their clinical conditions.
Although there is no accepted specific antiviral treatment regimen, 7 patients with pneumonia were treated with favipiravir. The decision to start favipiravir therapy was based on the clinical symptoms of the patients and the clinical experience of the medical team. The specific treatment protocol was defined as favipiravir (1.600 mg orally twice a day on day 1, then 600 mg orally twice a day for a total of 5 days) according to patient symptoms, hypoxemia, chest CT findings, and acute phase reactants (e.g., albumin, procalcitonin, C-reactive protein, fibrinogen, and ferritin). Antibiotics were used as needed, and the decision was based on the discretion of the patients' healthcare providers.

Statistical Analysis
All statistical analyses were performed using R v.3.6.0 (The R Foundation for Statistical Computing, Vienna, Austria; https ://ww w.r-p rojec t.org ). To assess the normality of the data, the Shapiro-Wilk normality test and Q-Q plots were used. Levene's test was also used to check the homogeneity of the variances. Numerical variables were presented as mean ± standard deviation or median (interquartile range: 25th-75th percentile) as appropriate. Additionally, categorical variables were described as number (n) and percentage (%). Fishe r-Fre eman-Halto n, Pearson chi-squared, Kruskal-Wallis, and Welch's F tests were run to determine whether there was a statistically significant difference or association between age groups according to the clinical characteristics and laboratory findings of the patients. Moreover, Yates's continuity correction chi-squared, Mann-Whitney, and Welch/independent sample t-tests were conducted to examine whether there was a statistically significant difference or association between patients with and without GI symptoms with demographic characteristics and laboratory examinations. Multiple comparisons of numerical variables found to be significant as a result of Kruskal-Wallis and Welch's F tests were performed with Bonferroni-adjusted Dunn and Games-Howell tests, respectively. Pairwise comparisons of categorical variables found to be significant as a result of Fishe r-Fre eman-Halto n or Pearson chi-squared tests were conducted alongside a 2-proportion Z-test. A 2-tailed P-value less than .05 was considered statistically significant.

Demographic and Clinical Characteristics of Children with COVID-19
A total of 294 patients (146 male, 148 female, median age 10 years [14 days to 18 years]) were enrolled in this study. Of these, 186 patients (63.2%) represented family cluster cases. Two neonates were born to mothers with COVID-19, and PCR tests were positive on days 3 and 7 after birth.
The clinical characteristics of patients according to age groups are given in Table 1. The most common symptoms were fever (71.1%), cough (39.5%), and diarrhea (15.6%). However, fever and vomiting were more common in children <5 years old; cough was more common in children older than 10 years (P = .001, P = 0.004, and P = .005, for fever, vomiting, and coughing, respectively). Although asymptomatic cases (11.7% vs. 4.1%) were more common in children under 5 years of age, pneumonia (4.8% vs. 0%) was common in children >10 years. The proportions of URTI, acute bronchitis, and pneumonia were 70.4%, 3.4%, and 2.7%, respectively. Two neonates and 7 obese adolescents with asthma presented with acute bronchitis. Two of 8 patients presenting with moderate pneumonia had immunosuppression, and the rest were obese adolescents with asthma. Bacterial coinfection (e.g., bacteremia or urinary tract) was detected in previously healthy patients (n = 6) under 5 years old. Three children in the 5-10-year group presented with acute appendicitis symptoms and reactive mesenteric lymph nodes.

Laboratory Characteristics and Liver Function Tests of Children with COVID-19
Demographic characteristics and laboratory examination data of the patients with GI manifestations are given in The laboratory results of patients with COVID-19 per age group are given in Table 3. Although the WBC, lymphocyte, and ESR values were significantly higher in children under 5 years of age than those aged 5-10 years and older than 10 years, there was no significant difference between children aged 5-10 years and older than 10. Children under 5 years had lower hemoglobin levels than children aged 5-10 years and children older than 10, whereas children aged 5-10 years had lower hemoglobin levels than children older than 10 years. The platelet levels were significantly higher in children under 5 years and 5-10 years than in children older than 10 (Table 3).
Abnormal ALP levels were significantly higher in children older than 10 years of age (12.2%) compared with children aged 5-10 years (1.9%) and under 5 years (0%). However, there was no significant difference between the abnormal ALP levels of children aged 5-10 years and children under 5. Bilirubin values (e.g., total, direct, and indirect) lower or higher than normal limits were not found to be significantly different between age groups (P = .920, P = .105, and P = .929, respectively).
High levels of ALT were reported in patients with 46.6% (n = 7) with 1-2 × ULN, 13.3% (n = 2) with 2-3 × ULN, and 40% Data were presented as mean ± standard deviation or median (interquartile range: 25th percentiles-75th percentiles), and were described as number (n) and percentage (%), as appropriate. 1 Independent samples t-test. 2 Yates continuity correction chi-square test. 3 Mann-Whitney U-test. 4 Welch's t-test. There is no consensus on the COVID-19 liver injury classifications. In an adult study, liver abnormalities were classified as hepatocellular (high AST/ALT activities), cholestatic (high ALP/ GGT activities), and mixed types. 16 In our study, based on the patterns of abnormal liver test results (n = 130, 44.2%), 71.5% (n = 93) were cholestatic, 18.4% (n = 24) were hepatocyte, and 10% (n = 13) were mixed. All abnormal liver result types were statistically significantly more frequent under 5 years of age (P < .001). No patient had liver failure at their followup evaluations as liver enzymes recovered within 5-60 days. All patient coagulation measures and kidney function results were normal. There were no differences in liver function tests (e.g., serum albumin level or INR) or clinical outcomes in groups with and without GI symptoms (P >.05). Only one adolescent with Canavan disease died due to acute respiratory distress syndrome. However, liver function tests were normal at admission, and AST and ALT levels increased following intensive care. The other patients were discharged from the hospital without complications.

DISCUSSION
In the early course of the COVID-19 pandemic, respiratory symptoms were the most frequently reported complaint in patients with SARS-CoV-2. Over time, patients were found to present with different clinical symptoms.
In our study, as with others, there was no gender or age dominance in COVID-19 patients.

Q2
Gastrointestinal and Liver Manifestations in Children with COVID-19 Although clinical findings in children tend to be mild, they may present with different systemic involvements. Guo's study indicated incidence rates of fever (77.9%), cough (32.4%), and diarrhea (4.4%). 17 Incidence rates of fever (40.5%), cough (5.9%), diarrhea (7.7%), and vomiting (4.1%) were reported by Cura et al. 18 In our study, the most asymptomatic cases included children under 5 years of age, and cough was more common among those >10 years (P < .001). The high rate of acute gastroenteritis in our study may be related to the frequency of COVID-19 testing in children who present with diarrhea.
Although children are less susceptible to COVID-19 than adults, underlying medical problems (e.g., genetic, neurological, or metabolic conditions; congenital heart disease; asthma; malignancies; obesity; and immunosuppression) and very young age are considered risk factors for severe cases. The most common comorbidities in our patients were asthma (n = 6), obesity (n = 7), autoimmune disease (n = 2: autoimmune hemolytic anemia and familial Mediterranean fever), malignancy (n = 2: hepatoblastoma and lymphoma), immunodeficiency (n = 2: nuclear factor-kappa B essential modulator deficiency and antibody deficiency), and Canavan disease (n = 1). These patients presented with acute bronchitis or pneumonia. All adolescents presenting with lower respiratory tract infections were obese and had asthma. A meta-analysis showed that the severity of COVID-19 was higher in children with asthma. 5,20 Öner et al 21 demonstrated that pediatric hematology-oncology patients have relatively favorable outcomes with COVID-19 because they are already under close monitoring. We observed that apart from asthma and obesity, immunosuppressive conditions and autoimmunities were risk factors.
In such cases, patients should be closely monitored as clinical deterioration may occur. However, there were no statistically significant elevations of liver enzymes in COVID-19 children with underlying diseases compared with previously healthy children (P >.05). Unlike adult patients, liver enzymes may not predict COVID-19 severity.
In our study, patients with bacterial coinfections (2%) were younger than 5 years of age and were previously healthy. All patients had high WBCs, CRP, and procalcitonin values and were discharged without complications. Although specific rates of pediatric patients have not yet been reported, bacterial infections were found to be more common in critically ill adult COVID-19 patients (6.9%). 22 There were no statistically significant differences in liver enzyme elevation and GI manifestations in the patient (P > .05). Procalcitonin was found to be high in 80% of Chinese pediatric patients, and 40% of these patients had coinfections. 23 High leukocyte, CRP, procalcitonin, and LDH levels were detected in 8 severe pediatric cases in China, and half of these had abnormal liver function test results. 24 In our study, WBC, CRP, and procalcitonin levels were high in immunosuppressed patients with pneumonia and those with bacterial coinfections. In others, there were no statistically significant differences between lymphopenia, liver enzyme elevation, or hospitalization rates (P > .05). Bacterial coinfections should be considered with COVID-19 patients who are immunosuppressive or with high acute phase reactants.
The notion that GI symptoms are prognostic indicators is controversial. In adults, GI symptoms and liver injuries have been associated with severe COVID-19. 25 However, another study found no differences in clinical outcomes in adults with or without GI symptoms. 2 In a meta-analysis, the prevalence of GI manifestations of COVID-19 in children was 32.5%. The most common symptoms are diarrhea, vomiting, and abdominal pain. 3 In our study, diarrhea was a symptom in all age groups, and it lasted for an average of 5 days. White blood cells, platelet count, and LDH levels were significantly higher in patients with GI symptoms. The results suggest that this may have been due to dehydration.
Although our patients with GI symptoms had good prognosis, we should be careful about symptoms that may progress in severity. Tullie et al 26 reported on 8 children with COVID-19 presenting with symptoms of atypical appendicitis. Terminal ileitis and mesenteric adenitis were later confirmed via radiological imaging, and no surgical intervention was required. However, the children's clinical status progressed to multisystemic inflammatory syndrome. In our study, 3 patients had mesenteric reactive lymphadenopathy via abdominal ultrasonography. Clinical signs of multisystem inflammatory syndrome in children or acute COVID-19 infection can mimic an acute surgical-like abdomen.
SARS-CoV-2 binds to the ACE-2 receptor and enters endothelial cells, some cholangiocytes, and centrilobular hepatocytes via the ACE-2 protein. 27 Abnormalities in liver tests have been reported in 14-53% of hospitalized patients. A meta-analysis has shown that liver injury may be associated with a severe form of COVID-19 in adults. 28 However, data on abnormal liver enzymes in children with COVID-19 are rare. Slight increases in ALT or AST have been reported in 15.6-29% of children. The underlying mechanisms of abnormal liver enzymes in pediatric patients are not as clear as in adults. The effects of ACE-2 receptors, antiviral drugs, and cytokine levels remain controversial in children. 29 In a meta-analysis of 551 laboratory-confirmed pediatric COVID-19 patients, 9% presented with increased ALT, and 18% exhibited high levels of AST. 3 Another meta-analysis suggested that the liver involvement prevalence was 1.9% among children. 30 In our study, the rates of AST, GGT, ALP, ALT, direct bilirubin, TBIL, and indirect bilirubin elevation were 33.3%, 8.9%, 6.6%, 5.1%, 3.8%, 3.8%, and 3.4%, respectively. The percentages of abnormal AST, ALT, and GGT were common in patients under 5 years of age. Like our findings, Zhou et al 29 suggested that COVID-19-related liver involvement is more prevalent among children aged 0-3 years than in other age groups as liver immaturity predisposes patients to a higher risk of abnormal