A Prospective Study of Clinical, Epidemiological and Serologic Status Features in a Paediatric Pcr Sars-Cov-2 Positive Population

Although most cases of SARS-CoV-2 infection in pediatric population are asymptomatic or mild, severe cases may occur in a small percentage. Unlike other respiratory infections, children usually develop it from infected adults. Percentage of seroconversión and outcome of antibodies after infection anti-SAR-CoV-2 in children are still poorly understood and may be different from adults. The rst aim was to describe clinical and epidemiological characteristics of pediatric patients diagnosed of SARS-CoV-2 infection in the second epidemic wave in two tertiary hospitals in Madrid, Spain and to determine the proportion of seronegative by comparing 3 serological techniques. As secondary aim it was proposed to determine the proportion of seronegative by comparing 3 serological techniques, seroconversion associated variables and the way of infection among the family members. A retrospective data collection was performed at 4-8 weeks after diagnosis on an outpatient basis, using a clinical-epidemiological questionnaire. At this time blood samples were collected to analyse the humoral response by 3 different serological assays. The quantitative variables are expressed as medians (interquartile range (IQR) 25-75). Univariate analysis of possible factors associated with seropositivity absence was performed. Results: 34-44) wave of the pandemic in Spain and to analyse the proportion of seronegative patients at 4 -8 weeks by 3 different serologic techniques. Other aims were the análisis of possible factors related to non-seroconversion, determine the concordance between the three serological techniques and the way of infection among the family members. Centaur ® XP SARS-CoV-2 Total (COV2T)); Abbott (Alinity® SARS-CoV-2 IgG II) and anti-IgG/A/M SARS-CoV-2 ELISA test (Human IgG/IgA/IgM anti-SARS-CoV-2 ELISA by The Binding Site Group Ltd., Birmingham, UK). The COV2T test (Siemens) is a chemiluminescent immunoassay (CLIA) for the qualitative detection of IgG and IgM antibodies against the spike protein (IgG-S and IgM-S) of SARS-CoV-2 in serum and plasma. This test uses a recombinant receptor-binding domain (RBD) antigen contained in the S1 subunit of the SARS-CoV-2 spike protein (S). Results are considered positive if index is ≥ 1 and negative if <1. The index value is established by two calibrators and ranges from 0.05 to 10.00. Abbott SARS-CoV-2 IgG II assay is a chemiluminescent microparticle immunoassay (CMIA). This test performs a qualitative determination of IgG antibodies against nucleocapsid protein (N) of SARS-CoV-2 (IgG-N) in serum and plasma. A result ≥ 1.4 is considered positive and negative when the index is <1.4. The anti-SARS-CoV-2 IgG / IgA / IgM assay is based on the determination of anti-RBD IgG / IgA / IgM antibody titers using the ELISA technique (enzyme-linked immunosorbent assay or immunosorbent assay linked to enzymes). Antibody titers are estimated by generating isotype-specic standard curves using monoclonal anti-SARS-CoV-2 IgG, IgA and / or IgM antibodies. Following the manufacturer's instructions positive samples were identied as those with a UR / mL three standard deviations above negative control samples mean. median and interquartile range (IQR). Statystical analysis between qualitative variables with seronegativity at 4-8 weeks was evaluated using chi-square test or Fisher's exact test. Overall agreement between the three tests was studied by calculating kappa index together with its 95% condence interval. A signicance value of 5% was accepted for all tests. Data processing and analysis was carried out using statistical package STATA v.15.0.

In univariate analysis a lower proportion of seroconversion was found in those children in whom COVID-19 con rmation is not documented among household contacts (15/29; 51.7%; p <0.05).

Conclusions:
Our results con rm the mild clinical picture in children during the second epidemic wave, and that in most cases, children are not the index case in the family. Likewise, in children the proportion in whom seroconversion is not detected is high, and seems higher than that reported in adults. In our series, an association of seronegativity was seen in the 2 to 10 year-old age group and when there was no documentation of COVID-19 in any household contact.
Background Page 3/15 The disease caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) was declared a global pandemic by OMS in March 2020 and it was designed as COVID-19. From the initial description it was patent the less severe disease in pediatric population [1][2][3] .
However a small proportion of children might develop a severe clinical course 4 or present as multisystem in ammatory syndrom related to SARS-CoV-2 (MIS-C) 5 . Although some studies show that the risk of infection in children is similar to adults 6 , others indicate that children may be less prone to infection 4,7,8 and that the prevalence is lower at early ages 9 .
In adult population it has been shown that the proportion of non-seroconverters is higher in paucisymptomatic patients or in the less severe cases [10][11][12] , being the most ill patients the ones with higher titers of antibodies 13 . Children tend to develop mild or even asymptomatic disease more often than adults [14][15] . This could lead to the possibility that the proportion of patients in whom no antibodies are detected is higher in the pediatric population as compared to adults 16,17,18 . There is evidence that the level of antibodies against the receptor -binding domain (RBD) of SARS-CoV-2´s Spike protein correlates with neutralizing antibodies which are of major importance in the protection against future infections. Nevertheless, kinetics of these two types of antibodies might not be the same 19 . Little is known about the evolution of antibodies against different antigens of SARS-CoV-2 in children, being the information yet scarce 20 . There is a decrease of the level of IgG antibodies after 6 months of infection in both children and adults 20 . Titers of neutralizing antibodies may stay high for longer periods of time specially in children aged 3 or less, even with higher levels found than in older children or adults 20 . The knowledge of the kinetics of the several antibodies produced after COVID-19 in children and its outcome could be of great importance in the protection against new infections and the response to vaccines.
The main aim of this study were to describe the clinical manifestations and epidemiological characteristics of SARS-CoV-2 in a pediatric population attended in two universitary hospitals in Madrid during the second wave of the pandemic in Spain and to analyse the proportion of seronegative patients at 4 -8 weeks by 3 different serologic techniques. Other aims were the análisis of possible factors related to non-seroconversion, determine the concordance between the three serological techniques and the way of infection among the family members.

Study design:
Multicenter prospective observational study includin children and adolescents who were attended at two universitary hospitals in Madrid (Hospital Clinico San Carlos and Hospital de Getafe) between September and November 2020, period corresponding to the second wave of the pandemic in Spain.

Study population:
All children and adolescents between 0 and 18 who were attended in the emergency room with SARS-CoV-2 infection con rmed were included.
Con rmed infection was considered when reverse transcription-polymerase chain reaction (RT-PCR) in a sample obtained from nasopharyngeal swab turned postive.
In the second wave, in order to increase detection of SARS-CoV-2 and to improve tracking contacts, a whole RT-PCR test was carried out by protocol in the Pediatric Emergency Department of both hospitals to all children who were attended for compatible symptoms. These included fever, respiratory, digestive or skin symptoms, as well as a history of recent close contact with a patient diagnosed with COVID-19 21 .
Those with con rmed infection were referred as outpatients to the clinic at 4-8 weeks, and were offered to participate in the present study, requiring to complete a clinical-epidemiological questionnaire and a blood sample, drawn for serology. Exclusion criteria were: immunosuppressed patients, rejecting to sign an informed consent, extraction of blood sample for serology outside the period of 4-8 weeks after a positive RT-PCR result, and not having available all 3 serological techniques results due to insu cient serum sample.
Data collection and study variables: The following variables were collected: demographic characteristics (age, sex, date of birth, place of birth, parental origin), relevant personal history, clinical manifestations at the time of going to the emergency room according to the corresponding medical report, as well as information collected in questionnaire at the rst consultation visit 4-8 weeks after diagnosis. The following clinical manifestations were collected: fever (≥38ºC), cough, dyspnea, gastrointestinal symptoms (abdominal pain, nausea, vomiting and / or diarrhea), skin lesions, neurological symptoms and other features.
Data related to diagnosis, treatment/procedures and outcome were also collected, both int he time of diagnosis and the follow-up.
A seropositive patient was de ned as the presence of humoral response in at least one of the 3 serological techniques and a seronegative patient if no humoral response was obtained in any of the 3 serological techniques.
The questionnaire also included questions about whether a family member or the child were the rst with appearing symptoms, so to de ne the index case. The index case was de ned as the subject with a con rmatory RT-PCR for SARS-CoV-2 infection and with an earlier onset date of symptoms in a speci c setting. Cases with symptoms onset shorter than 24 hours from the start date of the index case were considered co-primary cases. The subject in contact with an index case with a positive diagnostic test 24 hours or more after the date of the last positive test of the primary or co-primary case was de ned as a secondary case. When symptoms appeared 24 hours or more after the date of symptoms initiation of the primary or co-primary case was also de ned as a secondary case 22 .
The study was approved by the Ethics Committees of the two participating hospitals. Abbott SARS-CoV-2 IgG II assay is a chemiluminescent microparticle immunoassay (CMIA). This test performs a qualitative determination of IgG antibodies against nucleocapsid protein (N) of SARS-CoV-2 (IgG-N) in serum and plasma. A result ≥ 1.4 is considered positive and negative when the index is <1.4. The anti-SARS-CoV-2 IgG / IgA / IgM assay is based on the determination of anti-RBD IgG / IgA / IgM antibody titers using the ELISA technique (enzyme-linked immunosorbent assay or immunosorbent assay linked to enzymes). Antibody titers are estimated by generating isotype-speci c standard curves using monoclonal anti-SARS-CoV-2 IgG, IgA and / or IgM antibodies.
Following the manufacturer's instructions positive samples were identi ed as those with a UR / mL three standard deviations above negative control samples mean.

Statistic analysis:
Statistical analysis was performed out with STATA 15.0. Qualitative variables were presented with their absolute and relative frequency distribution. Quantitative variables were summarized with median and interquartile range (IQR). Statystical analysis between qualitative variables with seronegativity at 4-8 weeks was evaluated using chi-square test or Fisher's exact test. Overall agreement between the three tests was studied by calculating kappa index together with its 95% con dence interval. A signi cance value of 5% was accepted for all tests. Data processing and analysis was carried out using statistical package STATA v.15.0.

Discussion
Our study corroborate the mild disease in children during the second wave of the SARS-COV-2 pandemic in Spain 2,23,24 . In keeping with other paediatric series, the most commonly reported symptoms were fever followed by mild respiratory syndrome, headache and gastrointestinal disease 6 .
In a Spanish Paediatric Association review of COVID-19 manifestations in childhood, 18 studies were selected, mainly from Europe (52%) and mostly in patients admitted to hospital. Some differences were found compared to our group of patients. Fever followed by mild respiratory and gastrointestinal symptoms were the ones most commonly reported 25 . General as well as neurological symptoms were also reported, mainly fatigue, and headache, similar to our study. Dyspnoea was more common amongst the European group, and abdominal pain was the most frequent gastrointestinal manifestation 25 . Few children in our study required hospitalisation or speci c treatment. During the second wave of the pandemic a nasopharyngeal sample RT-PCR was performed per protocol to every child with symptoms consistent with COVID-19. This might explain why in our series a mild course of the disease was more common compared to the rst wave studies 24 . As previously reported in the literature, the index case at home was a child in less than 50% of cases, suggesting that adults are the main source of infection 7 .
In our study a large proportion of children were living with adults with symptomatic COVID-19 and adults with con rmed disease by RT-PCR.
The proportion of non seroconverters is high in our study with either the 3 serological techniques and seems to be higher than the gures reported in adulthood 11 .
Given the fact that the infection in childhood is frequently asymptomatic or paucisymptomatic it is possible that the proportion of individuals in whom no antibodies are detected is higher amongst the paediatric population as compared to the adult patients.
A progressive decay of antibodies titers is observed both in children and adults in the following months after the infection. The levels of antibodies vary alongside, anti-spike ones may be found earlier than anti nucleoproteins and the latter can also disappear faster 26 . However, it is possible that despite the absence of IgG antibodies against S protein and nucleoprotein, other neutralising antibodies might be produced. These ones may last longer and be responsible for a long-lasting protection against the disease. In adulthood this protection has been reported up to 15 months 18 .
Information about the length of antibodies titers against different SARS-CoV-2 antigens is scarce in childhood 20 and little is known about their progress in time. A decrease of IgG antibodies titers is observed six months after the infection both in children and adults 20 . However neutralising antibodies might stay high in the following months specially in children aged 3 or less, even with higher levels than the ones encountered in older children and in adults 20 . In a recent Italian study a persistence of neutralising antibodies is observed after 7-8 months of infection 20 . While IgG antinucleoprotein and anti-spike decrease over time and even disappear in up to 54% of patients 20 . In adults there is evidence of a correlation between antibodies against RBD of spike protein with neutralising antibodies. Nevertheless, the kinetics of these two types of antibodies might be different. 27 Both the presence of anti-spike and antinucleoprotein antibodies are associated with a lower risk of reinfection in the following 6 months 28 . The infection gives rise to an immune response in most cases however the protection against a reinfection is unclear. Immune response can be either humoral or celular and more studies are needed 26 .
It is possible that the variability in the kinetics of the different antibodies accounts for the slightly observed difference in sensitivity among the types of serologic tests used in our study. In any case, the proportion of non-seroconverter children is high and seemingly higher than the one reported in adults, although its signi cance in terms of protection remains to be elucidated.
In our series there is a statistically signi cant association between seronegativity and the age group 2-10 years, or being contact with a non-con rmed COVID-19 household. Although the signi cance of these ndings is uncertain, it could be related to the paucisymptomatology in this age group or due to a less exposure to the virus outside the familiar group. More studies are needed to determine factors involving the lack of seroconversion.
In terms of serology, we found a high concordance in the 3 tests used. Detection of antibodies using ELISA Human IgG/IgA/IgM anti SARS-CoV-2 yielded a higher sensitivity than with Abbot and Siemens techniques although the number of patients was small and therefore comparisons are not possible. Concordance between the 3 techniques adds value to our results on high grade of seronegativity in childhood as compared to adults 11 .
This study has somes limitations. The number of patients included is relatively small although it is related to the number of patients consecutively identi ed during the study period. It is a prospective analysis that may have missed some retrospective data obtained in a clinical and epidemiological questionnaire. Therefore, memory bias of parents and children may be present. Data were collected via questionnaires in an outpatient facility and some of them were not obtained in the precise moment the patients attended the emergency room. In some cases, we do not have accurate information about some symptoms such as loss of smell. The external validity of our study is limited because it only includes patients attended in the Emergency Department and may not be representative of the general population. Although SARSCoV2 diagnosis was based on a single positive PCR, due to the high prevalence at the time of the study (second wave of the pandemic in Spain), its positive predictive value is very high, in addition all patients had symptoms or an epidemiological situation that had a high clinical suspicion of infection that supports the positive PCR result However, our study has several strengths. On the one hand its prospective design, in the second wave of the pandemic in Spain with a selection according to pre-established criteria of consecutive patients with a RT-PCR con rmed diagnosis. And on the other hand, the serologic testing were performed at the same time lapse after the infection, and using different serologic testing, giving strength to our results.
In summary, our results offer additional data of the mild clinical picture in children, who usually are not the index case in the family. In addition, our series suggest that the proportion of children in whom seroconversion is not detected may be higher than in adults. More studies are needed to determine the factors involved in the humoral response and its signi cance in SARS-CoV-2 infected children.