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Marta Alenquer1, Tiago Milheiro Silva2, Onome Akpogheneta3, Filipe Ferreira1, Sílvia Vale-Costa1, Mónica Medina-Lopes1, Frederico Batista4, Ana Margarida Garcia2, Vasco M. Barreto5, Cathy Paulino6, João Costa6, João Sobral6, Maria Diniz-da-Costa6, Susana Ladeiro6, Rita Corte-Real7, José  Delgado Alves4,5, Ricardo B. Leite6, Jocelyne Demengeot3, Maria João Rocha Brito2, Maria João Amorim1

1 - Cell Biology of Viral Infection Lab, Instituto Gulbenkian de Ciência, Oeiras, Portugal,
2 - Pediatric Infectious Disease Unit, Hospital Dona Estefânia, Centro Hospitalar Universitário Lisboa Central, Lisboa, Portugal,
3 - Lymphocyte Physiology Lab, Instituto Gulbenkian de Ciência, Oeiras, Portugal,
4 - Department of Medicine Hospital Prof Doutor Fernando Fonseca, Amadora, Portugal,
5 - CEDOC NOVA, Centro de Estudos de Doenças Crónicas, Nova Medical School, Universidade Nova de Lisboa, Lisboa, Portugal,
6 - Genomics Unit, Instituto Gulbenkian de Ciência, Oeiras, Portugal,
7 - Molecular Biology Laboratory, Department of Clinical Pathology, Centro Hospitalar Universitário Lisboa Central, Lisboa, Portugal

- PLOS ONE | June 15, 2022

Background: Adults are being vaccinated against SARS-CoV-2 worldwide, but the longitudinal protection of these vaccines is uncertain, given the ongoing appearance of SARS-CoV-2 variants. Children remain largely unvaccinated and are susceptible to infection, with studies reporting that they actively transmit the virus even when asymptomatic, thus affecting the community.
Methods: We investigated if saliva is an effective sample for detecting SARS-CoV-2 RNA and antibodies in children, and associated viral RNA levels to infectivity. For that, we used a salivabased SARS-CoV-2 RT-qPCR test, preceded or not by RNA extraction, in 85 children aged 10 years and under, admitted to the hospital regardless of COVID-19 symptomatology. Amongst these, 29 (63.0%) presented at least one COVID-19 symptom, 46 (54.1%) were positive for SARS-CoV-2 infection, 28 (32.9%) were under the age of 1, and the mean (SD) age was 3.8 (3.4) years. Saliva samples were collected up to 48 h after a nasopharyngeal swab-RT-qPCR test.
Results: In children aged 10 years and under, the sensitivity, specificity, and accuracy of saliva-RTqPCR tests compared to NP swab-RT-qPCR were, respectively, 84.8% (71.8%–92.4%), 100% (91.0%–100%), and 91.8% (84.0%–96.6%) with RNA extraction, and 81.8% (68.0%– 90.5%), 100% (91.0%–100%), and 90.4% (82.1%–95.0%) without RNA extraction. Rescue of infectious particles from saliva was limited to CT values below 26. In addition, we found significant IgM positive responses to SARS-CoV-2 in children positive for SARS-CoV-2 by NP swab and negative by saliva compared to other groups, indicating late infection onset (>7–10 days).
Conclusions: Saliva is a suitable sample type for diagnosing children aged 10 years and under, including infants aged <1 year, even bypassing RNA extraction methods. Importantly, the detected viral RNA levels were significantly above the infectivity threshold in several samples. Further investigation is required to correlate SARS-CoV-2 RNA levels to viral transmission