key: cord-0956730-mpum0xmm
authors: Bahar, Burak; Jacquot, Cyril; Mo, Yunchuan D.; DeBiasi, Roberta L.; Campos, Joseph; Delaney, Meghan
title: Kinetics of viral clearance and antibody production across age groups in SARS-CoV-2 infected children
date: 2020-09-03
journal: J Pediatr
DOI: 10.1016/j.jpeds.2020.08.078
sha: 2ff73d0295b950cb687c4bd6d0f8f6a8f9842f03
doc_id: 956730
cord_uid: mpum0xmm

OBJECTIVES: To improve understanding of transition from viral infection to viral clearance, and antibody response in pediatric patients with SARS-CoV-2 infection. STUDY DESIGN: This retrospective analysis of children tested for SARS-CoV-2 by RT-PCR and IgG antibody at a quaternary-care, free-standing pediatric hospital between March 13, 2020 to June 21, 2020 included 6369 patients who underwent PCR testing and 215 patients who underwent antibody testing. During the initial study period, testing focused primarily on symptomatic children; the later study period included asymptomatic patients who underwent testing as preadmission or preprocedural screening. We report the proportion of positive and negative tests, time to viral clearance, and time to seropositivity. RESULTS: The rate of positivity varied over time due to viral circulation in the community and transition from targeted testing of symptomatic patients to more universal screening of hospitalized patients. Median duration of viral shedding (RT-PCR positivity) was 19.5 days and time from RT-PCR positivity to negativity was 25 days. Of note, patients aged 6 through 15 years demonstrated a longer time of RT-PCR positivity to negativity, compared with patients aged 16 through 22 years (median=32 versus 18 days, P = .015). Median time to seropositivity, by chemiluminescent testing, from RT-PCR positivity was 18 days while median time to reach adequate levels of neutralizing antibodies (defined as comparable to 160 titer by plaque reduction neutralization testing) was 36 days. CONCLUSIONS: The majority of patients demonstrated a prolonged period of viral shedding after infection with SARS CoV-2. It is unknown whether this correlates with persistent infectivity. Only 17 of 33 patients demonstrated adequate neutralizing antibodies during the timeframe of specimen collection. It remains unknown if IgG antibody against spike structured proteins correlates with immunity, and how long antibodies and potential protection persist.

Study design: This retrospective analysis of children tested for SARS-CoV-2 by RT-PCR and IgG antibody at a quaternary-care, free-standing pediatric hospital between March 13, 2020 to June 21, 2020 included 6369 patients who underwent PCR testing and 215 patients who underwent antibody testing. During the initial study period, testing focused primarily on symptomatic children; the later study period included asymptomatic patients who underwent testing as preadmission or preprocedural screening. We report the proportion of positive and negative . A pandemic was declared by the World Health Organization (WHO) on March 11, 2020 after the number of affected cases had increased significantly and the disease was observed in more than 100 countries. 2 SARS-CoV-2 has similar structural proteins present in other CoVs, consisting of spike (S), envelope (E), membrane (M), and nucleocapsid (N) components. 3, 4 Of these glycoproteins, virus-cell fusion of SARS-CoV-2 is mediated by the trimeric structure of the two functional subunits of the S protein, namely S1 and S2, after binding to angiotensin-converting enzyme 2 (ACE2). 5 Antibodies formed against the receptor binding domain (RBD) on the S1 subunit have the potential to neutralize SARS-CoV-2 by disabling virus-ACE2 binding and endocytosis. 6, 7 In addition, the competitive RBD binding capacity of these antibodies versus ACE2 correlates with neutralizing activity. 6 Dong et al reported epidemiologic results on 728 children with laboratory-confirmed COVID-19 from China. 8 The authors found that the median age was 7 years, >90% of the cases had a disease spectrum ranging from asymptomatic to moderate disease, and the proportion of severe and critical cases increased with age. 8 Subsequently, similar findings have been reported from Europe, the Middle East, and the US. [9] [10] [11] [12] J o u r n a l P r e -p r o o f Although there are emerging data regarding timing of viral clearance and immunologic response in adults with COVID-19 13 , there are few data in the pediatric population.

Furthermore, knowledge of factors affecting time-to-seropositivity is lacking in both pediatric and adult patients. 14 We report viral and antibody test results from our pediatric patient population in order to contribute to a better understanding of timing of viral clearance and antibody production in children with COVID-19. 

Antibody detection was performed from serum or plasma samples, collected in appropriate separator tubes, using DiaSorin Liaison XL SARS-CoV-2 IgG S1/S2 assay (DiaSorin, Saluggia, Italy).

We validated the analytical and clinical performance of this assay with similar outcomes as other researchers who previously reported satisfactory results. 16, 17 The test is based on J o u r n a l P r e -p r o o f chemiluminescent detection of antibodies against S1 and S2 glycoproteins of the virus using magnetic beads. Seropositivity was defined at presence of anti-SARS-CoV-2 IgG antibodies ≥ 15 absorbance units per milliliters (AU/mL), as recommended by the manufacturer. In addition to the qualitative results, quantitative test results were also included to reflect the amount of circulating IgG antibodies in patient samples at the time of blood draw. As demonstrated by the manufacturer, antibody results ≥ 80 AU/mL were comparable to a titer of 160 by plaque reduction neutralization testing (Liaison SARS-CoV-2 S1/S2 IgG (REF 311450)). According to these criteria, results were grouped as 'adequate for neutralization' and 'not adequate for neutralization', based on the 80 AU/mL threshold. In addition to routine testing ordered by clinical providers (n=194), we retrieved available leftover serum or plasma samples from patients who underwent RT-PCR testing but were not tested for antibodies (n=19). Serologic testing was also performed on these samples and their results were included in the present study.

Statistical analyses were performed using R software (R foundation for Statistical Computing, This project was undertaken as a quality improvement initiative at Children's National Hospital and therefore does not constitute human research. As such, it was not under the oversight of the institutional review board. This manuscript was evaluated and approved by the institutional publication review committee.

The total number of RT-PCR tests performed over the 100-day period was 7958 with 641 positive test results (Figure 1 ). Figure The median time from RT-PCR positivity to seropositivity (ie, antibody detection) was 18 days (95% CI=12-31) (Figure 4, A) . No difference in time to seropositivity was found between females (median = 18 days) and males (median = 21 days) (χ 2 =0.8, p=0.4) (Figure 4, B) . The median number of days for seroconversion from initial RT-PCR positivity was 29 days for the 0 through 5 age group, 11 days for the 6 through 15 age group, and 24 days for the 16 through 22 age group and overall comparison of age groups did not demonstrate a significant difference J o u r n a l P r e -p r o o f (χ 2 =1.6, p=0.4) (Figure 4 , C). After adjustment for sex, the various age groups also did not demonstrate significant differences in time to seropositivity (χ 2 =0.6, p=0.7). Only 17 of 33 patients demonstrated antibody levels ≥ 80 AU/mL, adequate levels of neutralizing antibodies as defined by the manufacturer, the median time to reach such a level in the 17 patients in this study was 36 days (95% CI=18-NA) ( Figure 5, A) . No significance was found for sex (χ 2 =1.1, p=0.3) (Figure 5, B) , age (χ 2 =0.9, p=0.6), or age stratified for sex (χ 2 =1.7, p=0.4) (Figure 5 , C).

In this study, we demonstrated that IgG class antibodies directed against S1 and S2 glycoproteins could be detected in blood samples of children before viral clearance. Previous studies revealed that antibodies bound to the RBD epitope of SARS-CoV-2's S1 glycoprotein are able of disrupting the virus-ACE2 interaction, thus blocking viral entry into human cells and demonstrating neutralizing capacity. 6 Although the RBD is located on the S1 subunit, the S2 subunit plays a crucial role in membrane fusion of the virus by conformation changes. 18, 19 It was previously hypothesized for SARS-CoV that multiple antibodies targeting different epitopes might act synergistically. 20, 21 As noted earlier, the antibody detection assay utilized in this study does not only measure antibodies targeting RBD but also includes all antibodies to epitopes on S1 and S2 glycoproteins. We propose that this assay design may be beneficial in assessing antibody response in individuals with a polyclonal immune response to both S1 and S2 antigens with synergistic antiviral activity. an increase in neutralization activity over time. 23 The authors reported that the variables associated with high neutralizing activity in a multivariate model included time from symptom onset to blood sampling, high BMI and male sex. 23 We demonstrated that females from 6 through 15 years had a longer period to viral clearance compared to other groups. This may be due to the age-dependent expression of ACE2 in the nasal epithelium, as demonstrated by Bunyavanich et al, where ACE2 gene expression was found to be significantly higher in older children (10 through 17 years) compared with younger children (< 10 years). 24 Furthermore, it was suggested that gonadal hormones play a role in ACE2 expression and function. 25 Taken together, increased duration of SARS-CoV-2 in the nasopharyngeal area could be an effect of hormonal changes in adolescent females in this age group. As noted by Chun et al, different sections of the airway feature variable expression of ACE2, and prolonged presence of the viral genome in the upper respiratory tract may not correlate with the severity of COVID-19. 26 A strength of our study was the inclusion of patients from multiple pediatric age groups with sequential PCR testing, which permitted comparison between age groups and sexes. One For COVID-19, antibody competition with ACE2, the intended target of SARS-CoV-2, and binding affinity of the antibody for RBD are critical to neutralization of the virus. 6 We demonstrated that the virus can be detected in nasopharyngeal samples with low levels of circulating antibody but becomes undetectable when levels reach neutralizing levels. This suggests that quantitative antibody results may be more useful for clinical management of patient. The U.S. Food and Drug Administration (FDA) has granted Emergency Use Authorization (EUA) to multiple virus detection and serologic tests for diagnosis and management of COVID-19. 15 However, serologic assays for SARS-CoV-2 are still in early phases of development. As of July 26, 2020, no commercial assay was cleared by FDA for quantitative reporting of the results. 15 In the present study, we showed that time to reach a quantitative result corresponding to a plaque reduction neutralizing antibody titer of 160 was associated with time to viral clearance. This titer cut point also has been recommended by the FDA to identify potential convalescent plasma donors.

However, it has been observed in the setting of other viral infections that seropositivity or antibody response may not correlate to immunity to the virus and that disease progression or J o u r n a l P r e -p r o o f reinfection is still possible. Tang et al reported that humoral immunity mounted against SARS-CoV gradually decreased over time and disappeared due to the lack of peripheral memory Blymphocyte response in most individuals. 30 This study has a number of limitations, which include its retrospective nature and timing of virus detection and antibody testing being at the discretion of the ordering provider rather than at defined time intervals. We have not included symptom onset in our analysis because this project was solely based on laboratory data evaluation. In addition, the serologic assay used in the study had 94.3% positive and 100% negative agreement with a comparative enzyme-linked immunosorbent assay. Given the low optimal positive agreement, some false-negative test results are expected.

Given the significant volume of testing performed, our study provides a timeline of viral clearance and humoral response to SARS-CoV-2 in pediatric patients, with through comparisons among age groups and sexes. We demonstrated that females 6 through 15 years of age had longer persistence of viral genome in nasopharyngeal samples. It should be noted that presence of viral genome may not correlate with transmissibility. Antibodies were detectable in low titer preceding viral clearance. The timing of antibodies reaching titers that correlate with potentially neutralizing levels coincided with RT-PCR negativity in nasopharyngeal samples within a 24 to 25 day period after initial RT-PCR positivity. However, only approximately 50% 

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We thank Eric Freeman and Celia Grant, MT(ASCP) for their efforts in this study.