Saliva molecular testing bypassing RNA extraction is suitable for monitoring and diagnosing SARS-CoV-2 infection in children
Authors: Alenquer Marta , Silva Tiago Milheiro , Akpogheneta Onome , Ferreira Filipe , Vale-Costa Sílvia , Medina-Lopes Mónica , Batista Frederico , Garcia Ana Margarida , Barreto Vasco M. , Paulino Cathy , Costa João , Sobral João , Diniz-da-Costa Maria , Ladeiro Susana , Delgado Alves José , Leite Ricardo B. , Demengeot Jocelyne , João Rocha Brito Maria , João Amorim Maria 
Published: August 2021

Structured abstractIMPORTANCEAdults 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 are susceptible to infection, and some studies reported that they actively transmit the virus even when asymptomatic, thus affecting the community. Methods to easily test infected children and track the virus they carry are in demand.OBJECTIVETo determine if saliva is an effective sample for detecting SARS-CoV-2 RNA and antibodies in children aged 10 years and under, and associate viral RNA levels to infectivity.DESIGN, SETTING, AND PARTICIPANTSIn this cross-sectional study, saliva SARS-CoV-2 RT-qPCR tests, with and without RNA extraction, were validated in 49 hospitalized adults. The test was then applied to 85 children, aged 10 years and under, admitted to the hospital regardless of COVID-19 symptomatology. Amongst 85 children, 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 positive test by nasopharyngeal (NP) swab-RT-qPCR.EXPOSUREInfection by SARS-COV-2 in adults up to 8 days post-symptom onset. Children admitted to hospital for any reason and therefore with unclear onset of SARS-CoV-2 infection.MAIN OUTCOMES AND MEASURESSaliva RT-qPCR up to CT<37 accurately identifies SARS-CoV-2 infected children, with viral infectivity in tissue culture restricted to CT<26.RESULTSIn adults, the accuracy of the saliva SARS-CoV-2 RT-qPCR test was 98.0% (95% confidence intervals [CI]: 89.3%–100%) as compared to NP-RT-qPCR. In children, the sensitivity, specificity, and accuracy of saliva-RT-qPCR 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. The threshold for rescuing infectious particles from saliva was CT<26. There were significant IgM positive responses to the spike protein and its receptor-binding domain (RBD) among 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 AND RELEVANCESaliva-molecular testing is suitable in 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 understand how SARS-CoV-2 RNA levels correlate with viral transmission.Key PointsQuestionIs saliva reverse transcription-quantitative polymerase chain reaction (RT-qPCR) testing (with and without RNA extraction) suitable to identify SARS-CoV-2 infected young children and can the cycle threshold (CT) be associated with infectivity in a heterogeneous population admitted to hospital for COVID-19-related and unrelated reasonsãFindingsIn this cross-sectional study of 85 children aged 10 years and under, RT-qPCR in saliva samples subjected or not to RNA extraction accurately detected SARS-CoV-2 RNA and infectious viruses could be recovered from CTs below 26.MeaningSaliva sampling coupled to RT-qPCR and specific antibody detection efficiently identifies infants and children infected with SARS-CoV-2. This approach is suitable for surveillance in kindergarten and school settings.