Shedding of infectious SARS-CoV-2 in symptomatic neonates, children and adolescents

Children are underrepresented in COVID-19 case numbers, with most pediatric cases exhibiting limited severity, and do not seem to be major drivers of transmission, unlike for other respiratory viruses. That said, SARS-CoV-2 infects children across all age groups, and despite the high proportion of mild or asymptomatic infections, it would be naive not to consider them as transmitters. To address this point we used cell culture to systematically assess the presence of cultivable SARS-CoV-2 in the upper respiratory tract in a cohort of our institution first 23 symptomatic neonates, children and teenagers with COVID-19 diagnosed by RT-PCR (See Appendix). Median age was 12.0 years (interquartile range [IQR 3.8-14.5], range 7 days-15.9 yrs). Most patients had an upper respiratory tract infection (n=13), followed by fever without source and pneumonia (each, n=2). Samples were collected at a median of 2 days (IQR 1-3) after symptom onset. Median viral load (VL) at time of diagnosis was 3.0x106 copies/ml (mean 4,4x108, IQR 6.9x103-4.4x108) from a nasopharyngeal swab (NPS). SARS-CoV-2 virus isolation was successful in 12/23 (52%) children after inoculating VeroE6 cells with a NPS specimen. SARS-CoV-2 isolation was determined by the presence of a typical cytopathic effect (CPE) and increased viral RNA in the supernatant. SARS-CoV-2 replication in all positive isolates (12/12) was confirmed by a second passage using new VeroE6 cells. Virus isolation was successful from NPS from all age groups, with a median initial VL of 1.7x108 copies/ml (mean 7.9x108, IQR 4.7x106-1.0x109) (Figure 1). The youngest patient that SARS-CoV-2 was isolated from was a 7-day old neonate. No correlation between disease presentation and success of virus isolation was observed. Our data show that initial VLs at diagnosis in symptomatic children is comparable to those in adults, and that symptomatic children of all ages shed infectious virus in early acute illness. Infectious virus isolation success was largely comparable to that of adults, although two specimens yielded an isolate at a lower VL (1.2x104 and 1.4x105 copies/ml) than what was observed in adults. SARS-CoV-2 shedding patterns of culture competent virus in symptomatic children resemble those observed in adults. Therefore, transmission of SARS-CoV-2 from children is plausible. Considering the relatively low frequency of infected children at this time, biological or other unknown factors could reduce transmission in this population. Both large serological investigations and systematic surveillance of acute respiratory diseases are needed to understand the role of children in this new pandemic.

SARS-CoV-2 was isolated from was a 7-day old neonate. No correlation between disease presentation and success of virus isolation was observed.
Our data show that initial VLs at diagnosis in symptomatic children is comparable to those in adults, and that symptomatic children of all ages shed infectious virus in early acute illness.
Infectious virus isolation success was largely comparable to that of adults, although two specimens yielded an isolate at a lower VL (1.2x104 and 1.4x105 copies/ml) than what was observed in adults.

Introduction
Children are underrepresented in COVID-19 case numbers1,2, with most pediatric cases exhibiting limited severity, and do not seem to be major drivers of transmission, unlike for other respiratory viruses3,4. That said, SARS-CoV-2 infects children across all age groups1,3, and despite the high proportion of mild or asymptomatic infections, it would be naïve not to consider them as transmitters. To address this point we used cell culture to systematically assess the presence of cultivable SARS-CoV-2 in the upper respiratory tract in a cohort of our institution's first 23 symptomatic neonates, children and teenagers with COVID-19 diagnosed by RT-PCR.
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Patients were either seen at the pediatric emergency room of the Geneva University Hospitals, or samples were received by the laboratory from other health care facilities as part of its function as the Swiss national reference laboratory for Emerging Viral Diseases at the Geneva University Hospitals.
All Nasopharyngeal (NPS) specimens were collected with a flocked swab in universal transport medium (both Copan, Italy) and tested for SARS-CoV-2 by according to manufacturers' instructions on various platforms, including initially in house method using eMAG extraction (bioMérieux, France) and Charité RT-PCR protocol6, then BD SARS-CoV-2 reagent kit for BD Max system (Becton, Dickinson and Co, US) and Cobas 6800 SARS For assessment of infectious virus, VeroE6 cells were seeded at a density of 8x104 cells/well in a 24 well plate and inoculated with 200 µl of viral transport medium the following day.
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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted May 1, 2020. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted May 1, 2020. Virus isolation was successful from NPS from all age groups, with a median initial VL of 1.7x108 copies/ml (mean 7.9x108, IQR 4.7x106-1.0x109) (Figure 1 and Supplementary Table   1). The youngest patient that SARS-CoV-2 was isolated from was a 7-day old neonate. No correlation between disease presentation and success of virus isolation was observed. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Discussion
Our data show that initial VLs at diagnosis in symptomatic children is comparable to those in adults8, and that symptomatic children of all ages shed infectious virus in early acute illness.
Infectious virus isolation success was largely comparable to that of adults, although two specimens yielded an isolate at a lower VL (1.2x104 and 1.4x105 copies/ml) than what was observed in adults8.
A limitation of our study was the small number of children assessed. Furthermore, the use of left-over material received for routine diagnostic purposes could have resulted in suboptimal times between sample collection and storage at -80°C due to transport and diagnostic processing time, resulting in a loss in infectivity and an underestimation of the initial number of viable viral particles. This might have led to a decreased titer of infectious virus and a failure of virus isolation even in the presence of high viral RNA levels. Of note, 2 of 3 samples with a high viral RNA level but unsuccessful virus isolation were collected outside our institution, and thus had longer transport times to the laboratory; the last one was collected in our institution, but the time between specimen collection and processing was 24 hours. The vast majority of patients were managed as outpatients and had to self-isolate at home after diagnosis, so no consecutive sampling was possible to assess infectious virus in multiple samples over the course of disease. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted May 1, 2020. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted May 1, 2020. SARS-CoV-2: severe acute respiratory syndrome coronavirus 2 All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted May 1, 2020.  (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.