key: cord-1045129-sp7c484t
authors: Isoldi, Sara; Mallardo, Saverio; Marcellino, Alessia; Bloise, Silvia; Dilillo, Anna; Iorfida, Donatella; Testa, Alessia; Del Giudice, Emanuela; Martucci, Vanessa; Sanseviero, Mariateresa; Barberi, Antonio; Raponi, Massimo; Ventriglia, Flavia; Lubrano, Riccardo
title: The comprehensive clinic, laboratory, and instrumental evaluation of children with COVID‐19: A 6‐months prospective study
date: 2021-02-16
journal: J Med Virol
DOI: 10.1002/jmv.26871
sha: 3afa857117a25a812dbb21013af89284a1499b64
doc_id: 1045129
cord_uid: sp7c484t

OBJECTIVES: To perform a comprehensive clinic, laboratory, and instrumental evaluation of children affected by coronavirus disease (COVID‐19). METHODS: Children with a positive result of nasopharyngeal swab for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) underwent laboratory tests, anal and conjunctival swab, electrocardiography, lung, abdomen, and cardiac ultrasound. Twenty‐four‐hour ambulatory blood pressure monitoring was performed if abnormal basal blood pressure. Patients were followed‐up for 6 months. RESULTS: Three hundred and sixteen children were evaluated; 15 were finally included. Confirmed family member SARS‐CoV‐2 infection was present in all. Twenty‐seven percent were asymptomatic. Anal and conjunctival swabs tests resulted negative in all. Patients with lower body mass index (BMI) presented significantly higher viral loads. Main laboratory abnormalities were: lactate dehydrogenase increasing (73%), low vitamin D levels (87%), hematuria (33%), proteinuria (26%), renal hyperfiltration (33%), and hypofiltration (13%). Two of the patients with hyperfiltration exhibited high blood pressure levels at diagnosis, and persistence of prehypertension at 6‐month follow‐up. No abnormalities were seen at ultrasound, excepting for one patient who exhibited B‐lines at lung sonography. Immunoglobulin G seroconversion was observed in all at 1‐month. CONCLUSIONS: Our study confirm that intra‐family transmission is important. The significant higher viral loads recorded among patients with lower BMI, together with low vitamin D levels, support the impact of nutritional status on immune system. Renal involvement is frequent even among children with mild COVID‐19, therefore prompt evaluation and identification of patients with reduced renal function reserve would allow a better stratification and management of patients. Seroconversion occurs also in asymptomatic children, with no differences in antibodies titer according to age, sex and clinical manifestations.

patients, 7 although data on clinic and radiologic evaluation are scarce in pediatrics. Moreover, the antibody responses against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain poorly understood and characterization and perdurance of seroconversion is unclear. 8 Aim of this prospective study was to perform a comprehensive clinic, laboratory and instrumental evaluation, including pulmonary, intestinal, renal and cardiac ultrasound (US) evaluation of children affected by COVID-19.

This is a prospective cohort study on pediatric patients (<18 years) with COVID-19 referred to the Pediatric Unit of Santa Maria Goretti Hospital, Latina-Sapienza University of Rome (Polo Pontino) for SARS-CoV-2 infection. All children admitted to our emergency department from April 6, 2020 to June 5, 2020 were evaluated for study inclusion. As per hospital protocol, to limit the contagion, every child was tested for SARS-CoV-2 infection at the admission, by using a nasopharyngeal swab for SARS-CoV-2 RNA. Children who resulted positive to the test and consented to the study protocol, were consecutively enrolled in the study.

At diagnosis, children underwent extensive laboratory tests, including serological test for COVID-19. A urine sample was collected for urine analysis. Twenty-four-hour urine collection was performed to evaluate kidney function. In addition, a fecal sample was collected for calprotectin level assessment. Patients underwent anal and conjunctival swab tests for SARS-CoV-2 nucleic acid assessment.

Moreover, 12-lead electrocardiogram (ECG), lung, abdomen and cardiac US was performed at baseline. Vital signs (oxygen saturation, blood pressure, pulse rate, and respiration rate) were monitored.

Twenty-four-hour ambulatory blood pressure monitoring (ABPM) was performed in patients with abnormal blood pressure. Prehypertension and hypertension were defined according to recent recommendations for the standard assessment of ABPM in children and adolescents. 9 Data on demographics, clinical history, comorbidities, family history of diseases, and SARS-CoV-2 infection were collected on an electronic database. Patients were followed-up for 6 months: visits were scheduled at 1 and 6 month. Urine analysis were repeated on a weekly basis, if abnormalities were seen. Similarly, nasopharyngeal swab test for SARS-CoV-2 was repeated on a weekly basis during the follow-up period until they were negativized. Hematological, kidney function test, and instrumental tests were repeated at 1 month if abnormal; further investigations or earlier repetition of abnormal exams were planned according to medical decision. Serology for COVID-19 was repeated at 1 and 6 months.

Written informed consent was obtained from parents of children or their legal surrogates before the enrollment. The study was explicitly approved by the institutional review board of Santa Maria Goretti Hospital. The work was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).

The presence of SARS-CoV-2 nucleic acid was detected on nasopharyngeal, anal, and conjunctival swab tests by real-time reversetranscription polymerase chain reaction (rRT-PCR) targeting three genes: envelope protein (E), RNA-dependent RNA polymerase (RdRp), and nucleocapsid protein (N).

The STARMag 96 × 4 Universal Cartridge Kit (Seegene Inc) was used to extract total RNA, and gene fragments were detected by Allplex 2019 n-CoV assay (Seegene Inc). The cycle threshold (C t ) values of rRT-PCR were used as indicators of the copy number of SARS-CoV-2 RNA. According to the manufacturer's instructions, samples with a C t value of <40 were regarded as SARS-CoV-2 detected, and a C t value <40 for only one of the three targets was considered positive.

Laboratory tests performed included: full blood count, clotting test (PT, aPTT, and D-dimers, fibrinogen, antithrombin III), blood gas analysis, immunoglobulins (IgA, IgM, and IgG), glucose, amylase, lipase, aspartate transaminase (AST), alanine transaminase, gammaglutamyltransferase, bilirubin, electrolytes, troponin, creatine phosphokinase, creatine kinase MB, lactate dehydrogenase (LDH), procalcitonin, serum ferritin, c-reactive protein (CRP), erythrocyte sedimentation rate (ESR), vitamin D, total proteins, albumin, urea, creatinine, lupus anticoagulant (LAC), antinuclear antibodies (ANA), extractable nuclear antigen (ENA), complement C 3 and C 4 , perinuclear antineutrophil cytoplasmic antibodies (p-ANCA), antineutrophil cytoplasmic antibodies (c-ANCA), anticardiolipin, anti-beta2 glycoprotein I (GPI) IgG and IgM, fecal calprotectin, urine analysis and 24 h urine collection for kidney function test, such as glomerular filtration rate (GFR), tubular phosphate reabsorption, proteinuria/creatininuria ratio (Pr/Cr), calcium/creatinine ratio (Ca/Cr), 24-h urine protein excretion (Prot/24 h). Vitamin D deficiency was defined if serum hydroxyvitamin D levels were less than 20 ng/ml, while insufficiency if between 20 and 30 ng/ml. 10 The iFlash-SARS-CoV-2 IgG and IgM assays (YHLO Biotech Co., Ltd) were used to evaluate the antibody response to the virus by chemiluminescence immunoassay, according to the manufacturer's instructions. The antibody levels were expressed as arbitrary unit per ml (AU/ml). The results ≥10 AU/ml was considered positive, and the results <10 AU/ml negative.

Patients underwent 12-lead ECG evaluation and a comprehensive US assessment, including lungs, heart, and abdomen, with a particular attention for kidneys and gut wall. To minimize interobserver variation, US were performed by operator experts in their field, specialized in acquiring 

Statistical analyses were executed using GraphPad Prism version 6 (GraphPad Software Inc.). Continuous variables were expressed as a median (range), median (interquartile range [IQR] ) and mean ± SD and categorical variables as frequencies (%).

We analyzed the differences between the groups using the χ 2 test for nominal variables. For continuous variables we tested the approximation to normal of the distribution of the population by Kolmogorov-Smirnov one-sample test and statistics for kurtosis and symmetry. As results were asymmetrically distributed, nonparametric tests were used. To analyze the potential association between the continuous variables (i.e., age, laboratory parameters) and nasopharyngeal swab positivity levels (C t ) a regression analysis was performed.

A p value below 0.05 was regarded as statistically significant. 

Confirmed family member SARS-CoV-2 infection was present in all the children included, and in particular 12 (80%) had both parents infected, and 3 (20%) only one, whose symptoms developed earlier. A total of 12 siblings were present in 11 families of the patients enrolled. The remaining four, were single child families. Siblings were all infected in 9/11 families (81.8%), with a total of 9/12 siblings (75%), and a negative result of rRT-PCR on nasopharyngeal swabs was described in 3/12 siblings (25%) from 2/11 (18.1%) different clusters.

Patients comorbidities were allergic rhinitis in 2 (13.3%), allergic asthma in 1 (6.7%) and coeliac disease in 1 (6.7%).

Characteristics of symptoms are described in Table 1 . Mean time from symptom onset and diagnosis was 2.45 days (±1.2 SD), with a median of 2 days (range = 1-5; interquantile range [IQR] = 1.5). Median temperature recorded among febrile patients was 38°C (mean = 37.7 ± 1.53; IQR = 1.5), with median fever duration of 3.6 days (mean = 1.9 ± 3.65; IQR = 2).

Physical examination revealed hyperemia of the pharynx in 8 (53.3%), abdominal swelling, tender to the touch in 5 (33.3%), active conjunctival injection with no discharge in 1 (6.7%). No abnormal breath sounds, or other signs were found. Vital signs were all within normal limits for age, excepting for 2 (13.3%) who exhibited a sisto-diastolic blood pressure greater than 90th percentile for age, sex, and height.

Characterization of genes detection of nasopharyngeal swab with mean C t values ± SD and 95% confidence interval (CI) are shown in Table 2 . The C t values were not influenced by the patients' age 

Main laboratory results are all summarized in Table 3 , while abnormal findings only are shown in Table 4 . No statistically significant differences in laboratory findings were observed among genders, different age groups, symptomatic and asymptomatic patients, and symptoms characteristics.

ECG was performed in all and showed no pathological signs. All patients underwent lung US, that was normal in all the patient 

After the initial evaluation, no patient presented sign or symptoms requiring hospitalization; therefore, they were managed with outpatient care with supportive therapy and home isolation instructions.

Regular exercise and low sodium diet were prescribed to those exhibiting elevated blood pressure levels. Ten out of eleven symptomatic patients (90.9%) reported symptoms resolution within a week of being diagnosed; 1/11 (9.1%) within 14 days. During follow-up, nasopharyngeal swabs were repeated on average every 7 days (mean 6.81 ± 3.7 days; range = 4-10). Table 2 shows nasopharyngeal swabs results at diagnosis and during follow-up period, with mean C t ± SD and 95% CI. The most common clinical manifestation in our cohort, although small, was fever, and that was in line with other pediatric reports. 15 Differently from literature data, we reported lower rates of respiratory symptoms, such as cough, that was present in 20% only of our cohort, compared to 40% in other reports. 20 reported lower median C t values among children <5 years compared to those 5-17 years or adults, 25 however nutritional status was not evaluated. A recent meta-analysis conducted in adults, 26 showed that higher BMI represented an important risk factor for complication, but with higher infection rates among patients with BMI < 25 kg/m 2 .

Study on children have reached mixed conclusions as to whether higher viral load is associated to severity of symptoms. 27, 28 Considering that our patients were all affected by a mild form of supporting the impact of nutritional status on immune system. Furthermore, low vitamin D levels have been found among infected patients, rising need for randomized controlled trials and large-scale cohort studies evaluating the association between vitamin D level and COVID-19 infection and severity. Renal involvement is frequent even among asymptomatic or mild COVID-19 pediatric patients, therefore prompt evaluation and identification of patients with reduced renal function reserve would allow a better stratification and management of patients. Even though we agree with Copetti, who defined the lung US "the stethoscope of the new millennium," 47 we did not find any significant abnormalities at lung, abdomen and cardiac sonography in children with mild disease. Lastly, we demonstrate that seroconversion occurs also in asymptomatic children, and no differences in antibodies titer was found between age, sex, and clinical manifestations. Larger study including more severe children with COVID-19 are needed to confirm and better interpret our findings.

The authors declare that there are no conflicts of interests.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Dr. Sara Isoldi conceptualized and designed this study, coordinated and supervised data collection, carried out the initial analyses, drafted the initial version of this paper, and reviewed and revised this paper. Formal analysis and investigations were performed by Dr. 

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Is lung ultrasound the stethoscope of the new millennium? Definitely yes