Why this paper is important to paediatric dentists? The COVID-19 has changed the approach of clinical activities in paediatric dentistry. To stablish the COVID-19 vigilance in paediatric population, it is important to include molecular tests for SARS-CoV-2 detection. Saliva is a less invasive and easy to handle sample for COVID-19 diagnosis. Our results have showed the same diagnostic performance of saliva compared to nasopharyngeal swabs in SARS-CoV-2 molecular detection in paediatric population.

Population-based testing is one of the most effective strategies for preventing COVID-19 transmission by allowing early identification of cases and decision-making based on the pandemic's behaviour.1 Nasopharyngeal swab (NPS) is the standard sample for molecular test, which causes much discomfort and possible cross-contamination during the collection.1, 2 Therefore, a trained healthcare professional is required for performing this procedure.1, 3 Saliva has been shown to be a reliable, safe diagnostic fluid for the detection of SARS-CoV-2, with a sensitivity ranging from 80%–100% compared with NPS in adult population.1, 3, 4 In paediatrics, the use of saliva has clear advantages because the collection of this fluid is less invasive, thus reducing the discomfort and allowing self-collection.2, 5 Few studies have been conducted for SARS-CoV-2 molecular detection by using saliva in children. A recent published study has shown sensitivity higher than 80% compared with NPS.6 The objective of this study was to compare the performance of saliva to that of NPS in the detection of SARS-CoV-2 in paediatric patients with mild symptoms.

This study evaluated saliva samples from children with suspected COVID-19 who attended public healthcare services of Araraquara, which is a medium-sized city located in the State of São Paulo, with a population of 238 339 in 2020.

As part of the COVID-19 contingency plan, the city of Araraquara offers molecular tests for the detection of SARS-COV-2 by using NPS in all symptomatic patients seeking healthcare service.

Parents and their children were invited to participate in this study at the time of NPS collection, in which the children were asked to spit into a sterile container for a collection of about 1 ml of saliva. The saliva samples were immediately stored at 4℃ until being taken to the laboratory (<48 h). Symptoms and delays between their onset and sample collection (days) were also recorded. Total RNA was extracted by using the viral RNA mini kit (Qiagen, GE). SARS-COV-2 detection was made by using the Altona RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (Altona Diagnostics GmbH), which employs a B-COV-specific probe directed to the E gene and a SARS-COV-2-specific probe directed to the S gene. Results were considered positive when one or both genes were amplified with a cycle threshold (Ct) <40. The positivity in saliva was later compared with the results of NPS obtained by the Araraquara health surveillance. Unfortunately, no information on viral load in the NPS test was available for further comparisons.

This study was approved by the Research Ethics Committee of the University of São Paulo School of Medicine under protocol number 4235245.

The sample consisted of 50 patients, in which 27 were girls (54%) and 23 were boys (46%). Ten were positive for SARS-CoV-2 in at least one sample collected (saliva or NPS). The mean age was 10.24 ± 3.52 years old, and saliva was collected after 4.76 ± 1.31 days from the symptoms. Of the 50 patients evaluated, symptoms were reported by 46 during the saliva collection and the main ones were the following: coryza (60.9%), cough (56.5%), sore throat (45.7%), headache (39.1%) and fever (30.4%). None of these symptoms was statistically associated with the diagnosis of COVID-19 (Table 1).

TABLE 1. List of symptoms reported Symptoms n % p a Fever Yes 14 30.4 .242 No 32 69.6 Cough Yes 26 56.5 .150 No 20 43.5 Shortness of air Yes 9 19.6 .384 No 37 80.4 Coryza Yes 28 60.9 .717 No 18 39.1 Headache Yes 18 39.1 .999 No 28 60.9 Myalgia Yes 7 15.2 .163 No 39 84.8 Fatigue Yes 8 17.4 .664 No 38 82.6 Nausea Yes 4 8.7 .201 No 42 91.3 Vomit Yes 4 8.7 .999 No 42 91.3 Diarrhoea Yes 6 13.0 .315 No 40 87.0 Abdominal pain Yes 8 17.4 .055 No 36 82.6 Loss of smell (anosmia) Yes 0 0 – No 46 100 Loss of taste (ageusia) Yes 2 4.3 .391 No 44 95.7 Sore throat Yes 21 45.7 .306 No 25 54.3

ROC curve analysis was performed in order to assess sensitivity and specificity of RT-PCR between saliva and NPS, with the positive cases considered a gold standard. The results showed a statistically significant curve for both samples, saliva and NPS, with same results (AUC = 0.900, SE = 0.076; p <.001; 95% CI = 0.750–1.00). With these results, we can state that saliva can be safely used for the diagnosis of COVID-19 in paediatric patients. We also tested the concordance between saliva and NPS by using Kappa concordance test (k = 0.702; p <.001), with 96% of the samples being concordant (Table 2). Additionally, the concordance between these fluids was assessed individually, in which positive cases (10/50 patients) were considered as true infections. It was observed that the saliva and NPS showed the same values for Kappa concordance test (k = 0.865, p <.001).

TABLE 2. Description of positive and negative cases of COVID-19 for saliva and NPS by using RT-PCR RT-PCR COVID-19 Total

Yes

N(%)

No

N(%)

Saliva Positive 8 (16) 0 (0) 8 (16) Negative 2 (4) 40 (80) 42 (84) Total 10 (20) 40 (80) 50 (100) NPS Positive 8 (16) 0 (0) 8 (16) Negative 2 (4) 40 (80) 42 (84) Total 10 (20) 40 (80) 50 (100) Note Sensitivity–80%; Specificity–100% for saliva and NPS; 95% CI (0.75–1.00). Abbreviation: NPS, Nasopharyngeal swabs.

Consistent scientific evidence has pointed to the effectiveness of the use of saliva as a diagnostic fluid for COVID-19 in adult population.1, 3, 4 The advantages of a less invasive and painless sample collection have been shown to be more evident in paediatric populations, which may include self-collection and multiple collection possibilities.2 There are, however, a few studies of paediatric patients and different ways to sampling for SARS-CoV-2 detection, and their results are conflicting.6-9 A recent study from Dubai has shown similar rates of SARS-CoV-2 detection in saliva and NPS by using RT-PCR. Sensitivity and specificity of saliva were 87.7% and 98.5% respectively.6 Our results showed that saliva had the same diagnostic performance as that of NPS for COVID-19, showing that this fluid is a good alternative for SARS-CoV-2 detection.

The use of less invasive strategies for COVID-19 surveillance has a crucial importance for children not only in the understanding of SARS-CoV-2 behaviour in this population, but also in the re-opening of schools based on constant tracking of asymptomatic cases.2, 6 Additionally, with the emergence of the variants of concern and vaccination campaigns that were initially not aimed at this age group, saliva could help on the detection of them in paediatric patients. The use of saliva makes COVID-19 surveillance viable, since this strategy is more largely accepted by individuals for being painless and for requiring no professional sample collection, avoiding possible technical problems on performing NPS, especially in paediatric population.2-6 Depending on the age group, the individuals themselves can be remotely instructed (eg, videos) to perform the sample collection.2, 3

The limitations of this preliminary work are in the fact that larger samples and inclusion of asymptomatic children should be considered in further studies. Our data allow us to conclude that saliva is a viable alternative fluid for the molecular diagnosis of COVID-19 in children.

ACKNOWLEDGMENTS

The authors would like to thank José Tadeu Sales for the language correction of the manuscript. This study was supported by the São Paulo Research Foundation (FAPESP) according to grant # 2019/03859-9.

AUTHOR CONTRIBUTIONS

Alvina C. Felix, Walter M. Figueiredo and Dmitry J. S. Sarmento conceptualized and designed the study, drafted the initial manuscript and reviewed and revised the manuscript. Anderson V. de Paula, Andreia C. Ribeiro, Francini C. da Silva, Marta Inemami, Angela A. Costa, Cibele O. D. Leal and Tatiana A. Sassaki designed the data collection instruments, collected data, carried out the initial analyses and reviewed and revised the manuscript. Claudio S. Pannuti, Paulo H. Braz-Silva and Camila M. Romano conceptualized and designed the study, coordinated and supervised the data collection, draft the manuscript and critically reviewed the manuscript for important intellectual content. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.