Particularities of the Evolution of SARS-CoV-2 Infection in Children

SARS-CoV-2 virus infection was rst reported in China in late 2019 and has spread rapidly around the world. There is little information about the peculiarities of COVID-19 infection in children because the number of infected children was small, around 2% of all diseases. In this retrospective study, we recruited 143 children infected with SARS-CoV-2 between March and October 2020, in Sibiu, Romania. RT-PCR tests, serum SARS-CoV-2 IgG/ IgM antibodies, lung radiography, biochemical and hematological tests were performed during the hospitalization. Of the 143 children selected in the study, 47.0% were male and 53% were female. At admission, all children tested positive for SARS-CoV-2, collecting nasopharyngeal exudate. Clinical manifestations included: cough in 75.52% of cases, fever in 55.94% of cases, nasal obstruction in 50.34% of cases, rhinorrhea in 38.46% of cases, muscle pain in 26.57% of cases, fatigue in 17.48% of cases, diarrhea and headache in 14.68% of cases. In 21 children (14,68%), the number of leukocytes was increased. In 38 cases (26,57%), the lung radiograph showed changes similar to bronchopneumonia, and the other cases did not have pulmonary changes. The persistence of the virus in the body of infected children is above the average reported in studies performed in adults, the virus being identied in the respiratory tract between 16 and 34 days. IgG class antibodies in patients' serum appeared between the 4th day of hospitalization and up to a maximum of 25 days, with a mean of 16.5 days. The of the virus in the body of infected children is above the average reported in studies performed in the virus being identied in the respiratory tract between 16 and 34 days. IgG class antibodies in patients' appeared within All

The virus can be transmitted by an infected person or an asymptomatic carrier and is an extremely contagious disease. The main route of transmission of the virus is the respiratory tract, through the Flügge droplets [12]. The incubation period is between 5 and 14 days [1]. The spread of the virus was rapid [13], so that the infection with the new coronavirus generated many cases of pneumonia worldwide.
Symptomatology of the disease is predominantly respiratory (fever, cough, di culty breathing) [14,15], the vast majority of cases with symptoms of minimal and moderate intensity and only in about 20% of cases may occur severe manifestations (bilateral interstitial pneumonia), with evolution to respiratory failure and acute respiratory distress (ARDS) [16]. There are also cases with gastrointestinal manifestations (especially diarrhea), and in some patients, hypo / anosmia (loss of smell) and hypo / dysgeusia (loss of taste) have been reported as early symptoms.
The reference test for the speci c diagnosis of COVID-19 is a molecular biology test, performed by NAAT technology (nucleic acid ampli cation technology), respectively, reverse transcription real time PCR (RT-PCR) that detect the presence of viral RNA in the nasopharynx [13]. In order to show the body's response to infection, serological tests are performed that detect the presence of antivirus antibodies (SARS-CoV-2) [17,18], these antibodies being immunoglobulins belonging to classes A, M, G, less often D.
Tests that identify the virus should be performed taking into account the incubation period of the virus in the host organism, respectively after a period of 5-14 days. To identify the body's serological response, models from other viral infections should be considered, which generally have the following sequence of antibodies: IgA antibodies, closely followed by IgM, and nally IgG-type antibodies. which remain in high titers for a longer period of time [19].
Currently, the body's speci c immune response to SARS-CoV-2 infection is the subject of numerous studies in all countries. To date, there is no complete validated information on the dynamics of antibody onset of infection and their persistence after SARS-CoV-2 virus clearance.
Our study wants to analyze the persistence period of the virus in the body of children identi ed positively with SARS-CoV-2 virus mas well as the peculiarities of their immune response.

Materials And Methods
The study included 143 patients, aged between 17 days and 18 years, hospitalized in the COVID Infectious Diseases Department of the Pediatric Clinical Hospital in Sibiu between March and October 2020. This series of cases was approved by the institutional ethics commission (no. 4065/12.06.2020).
Demographic information was selected, including age and gender.
Patients underwent RT-PCR tests to identify the presence of the virus in the respiratory tract by collecting a nasal exudate and a pharyngeal exudate on the rst day of hospitalization, and later on days 12-14 to see if there was persistence of the virus for discharge patient after the existence of 2 negative RT-PCR tests.
On day 12 after admission, patients' blood was collected to highlight the body's immune response by determining IgM/IgG antibodies by a rapid immunochromatographic test for the simultaneous detection of IgM/IgG antibodies in human blood (serum/plasma).
For RT-PCR testing, automatic extraction is used, on a 24-position extractor Lab-Aid 824 -Zeesan, and the ampli cation is done on a BIORAD CFX2s device. Other biochemical and hematological markers were investigated to assess the severity of the disease, as well as radiological images to highlight pneumonia.
For statistical analysis, categorical variables were expressed as number (%) while continuous variables were expressed as median (IQR -interquartile range). For laboratory measurements results are presented for both quantitative and qualitative (inside and outside normal ranges) versions. We used R version 4.0.1 (R Foundation for Statistical Computing; http://www.r-project.org/) for all analyses.
During the hospitalization of the patients, an average of 7 tests were performed (with a variation of 6 -10 tests / patient). The mean time from RT-PCR positive to RT-PCR negative was 26.5 days (IQR 6 -34 days). IgG and IgM antibodies for serum SARS-CoV-2 were assayed, with antibodies averaging 11.5 days (IQR 1-17 days). All patients had unchanged oxygen saturation (Table 1).
After the appearance of IgG-type antibodies, all patients remained under observation due to the positive results of the median RT PCR COVID test -19 was 16.5 days (IQR 4 -25 days).
Laboratory results are as follows: the number of leukocytes was normal in 114 cases (79.72%),  Table 3).
The values of the continuous variables were standardized (using z scores). We rescale the original variables to have equal range and/or variance for the variables in the graphic (the initial variables were measured at different scales). (Fig. 2)

Discussion
On 5 September 2020, there have been more than 29 million cases worldwide with more than 929,000 deaths [20] and the amount of children with COVID-19 is under 2% of all cases [21].
In China, the average age of infected children was 10.9 years, with the highest incidence in children between 5 and 14 years. The incidence between the sexes was slightly higher among females. The most common symptom, for which the child went to the doctor was cough, which was found in 73.52% of cases, other common symptoms were: fever, reported in 55.94% of cases, nasal obstruction in 50.34%, rhinorrhea in 38.46%, muscle pain in 26.57% of cases, diarrhea in 14.68% of cases, data that are consistent with the 2020 study by ZHENG F et al. [22].
Loss of smell and taste was a rare symptom in children, being recorded in 5.59% of cases, in accordance with the 2020 study performed by Luethgen M, et al., these symptoms meeting in the 5,18% of cases of SARS-CoV-2 [23].
In Romania, from the end of February 2020, when the rst positive patient appeared, the number of cases was 164477. In the Sibiu area the total number of diseases was, until October 14, 4016 patients, of which 143 children under 18 years, representing a percentage of 3.56% of the total cases.
Considering the hemoleucogram, we noticed a decrease in lymphocytes in 61.53% of cases, compared to the study conducted by Xia et al. 2020 [1] where a percentage of 35% was reported, and C-reactive protein was increased in 23.77% of cases compared to the same study, in which C-reactive protein was modi ed in 45% of cases. Unilateral and bilateral pneumonia is much lower in children included in the present study 11.8%, respectively 14.69%, compared to studies conducted in 2020 by Zeng F, where unilateral pneumonia was found in 30%, respectively 20.8% of cases, and bilateral pneumonia in 50%, respectively 45.8% of cases [22].
Pediatric patients had a good prognosis with an average hospitalization of 26.5 days, the number of hospitalization days being high compared to other studies, in which the number of hospitalization days was lower, respectively 12.9 days [1].
During the hospitalization, a large number of tests were performed to identify the appearance of antibodies and the disappearance of viral RNA, the average of the tests performed being 7 (with a variation of 6 -10 tests / patient). The mean time from RT-PCR positive to RT-PCR negative was 26.5 days, consistent with the study conducted by Bahar B. [24].
The mean time between the positive PCR-RT test and the appearance of serum IgG antibodies in the serum was 11.5 days, a lower average compared to the study conducted by Bahar B. in which this average time was 18 days.
The current study showed that IgG antibodies are detected in children's serum samples before the virus is eliminated from the body, the antibodies being present at the same time as the presence of the virus in the body.
After the appearance of IgG antibodies in the serum, until the negation of the RT-PCR test, the virus was present in the body for another average period of 16,5 days. Oxygen saturation was unchanged for all pediatric patients.
The children were discharged after presenting two negative RT-PCR COVID -19 tests at an interval of 2 days.
The most obvious symptoms in children were cough and fever in concordance with the study of Martelletti L. et al. [25], unlike adults where fever, fatigue, loss of smell and taste were the most important symptoms. All children included in the study developed mild forms of the disease, in accordance with the literature [26,27].
The persistence of the virus in the body of infected children is above the average reported in studies performed in adults, the virus being identi ed in the respiratory tract between 16 and 34 days.
IgG class antibodies in patients' serum appeared between the 4th day of hospitalization and up to a maximum of 25 days, with a mean of 16.5 days. IgG antibodies exist simultaneously with the presence of the virus, for a long time, different from the general model for other types of viral infections, which determines the need to identify not only antibodies at discharge but to highlight the absence of virus by RT-PCR test.
The long period of hospitalization required for children until the infection was negated generated high costs / patient and a sustained effort on the part of the medical team that had to manage the isolation problems caused by hospitalization in the limited space of the hospital ward.
To date, there is no speci c treatment to reduce the rates of SARS-CoV-2 infection [28] but recent research has indicated that an optimal level of vitamin D in the blood may be an important factor in reducing SARS-CoV-2 infection. Vitamin D supplementation is bene cial for solving the public health problem of counteracting the general vitamin D de ciency among the pediatric population [29].