Saliva: an important alternative for screening and monitoring of COVID-19 in children

Catielma Nascimento Santos Karla Mayra Rezende Nilson Ferreira de Oliveira Neto Thelma Suely Okay Paulo Henrique Braz-Silva Marcelo Bönecker About the authors

Abstract:

The increasing number of cases of COVID-19 worldwide poses challenges to healthcare systems not only in effectively identifying individuals positive for SARS-CoV-2, but also in isolating cases to minimise contagion in early diagnosing more severe cases that will need hospitalization. Less-invasive collection methods are indispensable in a pandemic scenario as large-scale tests are necessary to understand the actual evolution of contagion in different populations, thus enabling decision-making based on scientific evidence. Saliva has been shown to be an alternative for diagnosing viral infections as this biological fluid can be easily and quickly collected without using specific devices and causing less discomfort during collection, which is an important factor for use in children. Despite the smaller percentage of severe cases of COVID-19 among children, they seem to play an important role in the contagion as they have the same potential of transmission as that of adults. Knowing the evolution of COVID-19 pandemic in children is extremely important, mainly regarding the changing in rules of social distancing, such as re-opening schools and recreational activities spaces. In addition, countless cases of a severe multi-systemic inflammatory syndrome that shares clinical and laboratory features with Kawasaki's disease have been recently related to SARS-CoV-2 infections in children, adolescents and young adults. In view of this scenario, the aim of this study was to present saliva as an alternative for seeking diagnostic and prognostic markers of COVID-19 in children, including adequate sample collection techniques for different age groups.

Keywords:
Coronavirus; COVID-19; Diagnosis; Pediatrics; Saliva

Introduction

An epidemic of the new coronavirus started in the city of Wuhan, Hubei Province, China, in early December 2019.11. Chan JF, Yuan S, Kok KH, To KK, Chu H, Yang J, et al. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet. 2020 Feb;395(10223):514-23. https://doi.org/10.1016/S0140-6736(20)30154-9
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The coronavirus-associated disease (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that spread very quickly worldwide, so that COVID-19 was declared a pandemic by the World Health Organization (WHO) in March, 2020. Coronaviruses are known to be pathogenic to both, animals and humans, and are related to gastrointestinal and respiratory symptoms.22. Salata C, Calistri A, Parolin C, Palù G. Coronaviruses: a paradigm of new emerging zoonotic diseases. Pathog Dis. 2019 Dec;77(9):77. https://doi.org/10.1093/femspd/ftaa006
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The disease may be symptomatic or asyntomatic.33. Patel NA. Pediatric COVID-19: systematic review of the literature. Am J Otolaryngol. 2020 Sep - Oct;41(5):102573. https://doi.org/10.1016/j.amjoto.2020.102573
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The children appear to have milder symptoms than adults.44. Mallineni SK, Innes NP, Raggio DP, Araujo MP, Robertson MD, Jayaraman J. Coronavirus disease (COVID-19): characteristics in children and considerations for dentists providing their care. Int J Paediatr Dent. 2020 May;30(3):245-50. https://doi.org/10.1111/ipd.12653
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However, the course of the disease is worse and can lead to death, especially in risk groups such as the elderly and individuals with hypertension, diabetes, obesity, chronic respiratory and cardiovascular diseases.55. Yang J, Zheng Y, Gou X, Pu K, Chen Z, Guo Q, et al. Prevalence of comorbidities and its effects in patients infected with SARS-CoV-2: a systematic review and meta-analysis. Int J Infect Dis. 2020 May;94:91-5. https://doi.org/10.1016/j.ijid.2020.03.017
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However, there are a few cases reporting children who died due to COVID-19.66. Dong Y, Mo X, Hu Y, Qi X, Jiang F, Jiang Z, et al. Epidemiology of COVID-19 Among Children in China. Pediatrics. 2020 Jun;145(6):145. https://doi.org/10.1542/peds.2020-0702
https://doi.org/10.1542/peds.2020-0702...

Over time and the pandemic spreading to different countries and continents, the number of cases of COVID-19 among children is increasing and the cases of Kawasaki's-like disease have also been more frequently reported.77. Dasgupta K, Finch SE. A case of Pediatric Multisystem Inflammatory Syndrome temporally associated with COVID-19 in South Dakota. S D Med. 2020 Jun;73(6):246-51. The role of children in the COVID-19 transmission remains unclear, but there are no reports indicating that children have less potential of dissemination than adults.88. Jones VG, Mills M, Suarez D, Hogan CA, Yeh D, Segal JB, et al. COVID-19 and Kawasaki disease: novel virus and novel case. Hosp Pediatr. 2020 Jun;10(6):537-40. https://doi.org/10.1542/hpeds.2020-0123
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,99. Hong KH, Lee SW, Kim TS, Huh HJ, Lee J, Kim SY, et al. Guidelines for Laboratory Diagnosis of Coronavirus Disease 2019 (COVID-19) in Korea. Ann Lab Med. 2020 Sep;40(5):351-60. https://doi.org/10.3343/alm.2020.40.5.351
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That is why diagnostic testing in this group is so relevant, particularly regarding the re-opening of kindergartens and schools as children have a poorer understanding of biosecurity measures.

Nasopharyngeal, oropharyngeal and related-nasopharyngeal aspiration techniques may be used for diagnosing the new coronavirus. In hospitalized patients, lower respiratory tract specimens such as sputum, broncho-alveolar or tracheal lavage and aspirates can be used to perform the gold standard diagnostic technique, the Real Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR).88. Jones VG, Mills M, Suarez D, Hogan CA, Yeh D, Segal JB, et al. COVID-19 and Kawasaki disease: novel virus and novel case. Hosp Pediatr. 2020 Jun;10(6):537-40. https://doi.org/10.1542/hpeds.2020-0123
https://doi.org/10.1542/hpeds.2020-0123...
Aside for the high cost of reagents and dedicated equipment, the collection and processing of samples are time consuming and require specialized handlers to perform the sample collection through the insertion of swabs at the deepest point of the oropharynx and nasopharynx, frequently causing discomfort and retching, especially in children. As a good alternative, saliva has been shown to be an effective biological sample for carrying out viral detection methods99. Hong KH, Lee SW, Kim TS, Huh HJ, Lee J, Kim SY, et al. Guidelines for Laboratory Diagnosis of Coronavirus Disease 2019 (COVID-19) in Korea. Ann Lab Med. 2020 Sep;40(5):351-60. https://doi.org/10.3343/alm.2020.40.5.351
https://doi.org/10.3343/alm.2020.40.5.35...
and a less traumatic/invasive procedure for pediatric patients.1010. Granger DA, Kivlighan KT, Fortunato C, Harmon AG, Hibel LC, Schwartz EB, et al. Integration of salivary biomarkers into developmental and behaviorally-oriented research: problems and solutions for collecting specimens. Physiol Behav. 2007 Nov;92(4):583-90. https://doi.org/10.1016/j.physbeh.2007.05.004
https://doi.org/10.1016/j.physbeh.2007.0...

As the pandemic progresses towards the stabilization in some countries, there is an imminent need to re-open kindergartens, schools, as well as recreational spaces for children. However, less complex collection methods for the identification of SARS-CoV-2 are necessary, mainly through large-scale testing, in the case of children in schools. Saliva is a good biological fluid because it enables the identification of different viruses, as has already been shown in other infectious models.1010. Granger DA, Kivlighan KT, Fortunato C, Harmon AG, Hibel LC, Schwartz EB, et al. Integration of salivary biomarkers into developmental and behaviorally-oriented research: problems and solutions for collecting specimens. Physiol Behav. 2007 Nov;92(4):583-90. https://doi.org/10.1016/j.physbeh.2007.05.004
https://doi.org/10.1016/j.physbeh.2007.0...
Furthermore, saliva can be quickly and conveniently collected by using cheap and non-invasive methods.1010. Granger DA, Kivlighan KT, Fortunato C, Harmon AG, Hibel LC, Schwartz EB, et al. Integration of salivary biomarkers into developmental and behaviorally-oriented research: problems and solutions for collecting specimens. Physiol Behav. 2007 Nov;92(4):583-90. https://doi.org/10.1016/j.physbeh.2007.05.004
https://doi.org/10.1016/j.physbeh.2007.0...
For younger children, who seem to be one of the target audiences in the context of saliva sampling instead of respiratory secretions, a saliva test is a much more attractive and less invasive form of collection, and this paper describes a number of saliva collection techniques for children in different age groups. There is no specific information in the literature on the types of test and how to perform salivary for the diagnosis of COVID-19. Therefore, the aim of this study was to review the literature on the possibilities to diagnose COVID-19 in children by means of saliva sampling and analyses and to describe appropriate collection techniques for different age groups.

Symptomatology of COVID-19 in childhood

There are a few studies on COVID-19 in children. When compared to infected adults, the number of cases is lower in children, symptoms are considered milder, the mortality rate is lower1111. Ludvigsson JF. Systematic review of COVID-19 in children shows milder cases and a better prognosis than adults. Acta Paediatr. 2020 Jun;109(6):1088-95. https://doi.org/10.1111/apa.15270
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,1212. Yung CF, Kam KQ, Chong CY, Nadua KD, Li J, Tan NW, et al. Household transmission of Severe Acute Respiratory Syndrome Coronavirus 2 from adults to children. J Pediatr. 2020 Oct;225:249-51. https://doi.org/10.1016/j.jpeds.2020.07.009
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and the prognosis is more favorable.33. Patel NA. Pediatric COVID-19: systematic review of the literature. Am J Otolaryngol. 2020 Sep - Oct;41(5):102573. https://doi.org/10.1016/j.amjoto.2020.102573
https://doi.org/10.1016/j.amjoto.2020.10...
,1313. Shen KL, Yang YH, Jiang RM, Wang TY, Zhao DC, Jiang Y, et al. Updated diagnosis, treatment and prevention of COVID-19 in children: experts’ consensus statement (condensed version of the second edition). World J Pediatr.2020;16:232-9. https://doi.org/10.1007/s12519-020-00362-4
https://doi.org/10.1007/s12519-020-00362...
It has been shown that in infants and young children, the disease is relatively more severe than in older children and adolescents.1414. Sankar J, Dhochak N, Kabra SK, Lodha R. COVID-19 in Children: Clinical Approach and Management. Indian J Pediatr. 2020 Jun;87(6):433-42. https://doi.org/10.1007/s12098-020-03292-1
https://doi.org/10.1007/s12098-020-03292...

At the present time, the contamination in this group occurs through contact with infected adults, as children's environments such as schools, kindergartens and playgrounds remain closed.88. Jones VG, Mills M, Suarez D, Hogan CA, Yeh D, Segal JB, et al. COVID-19 and Kawasaki disease: novel virus and novel case. Hosp Pediatr. 2020 Jun;10(6):537-40. https://doi.org/10.1542/hpeds.2020-0123
https://doi.org/10.1542/hpeds.2020-0123...
There is a low probability of transmission to neonates whose mothers are diagnosed with COVID-19 at delivery, provided that breastfeeding is performed with the recommended precaution measures, such as the correct washing of hands and use of face mask by the mother.33. Patel NA. Pediatric COVID-19: systematic review of the literature. Am J Otolaryngol. 2020 Sep - Oct;41(5):102573. https://doi.org/10.1016/j.amjoto.2020.102573
https://doi.org/10.1016/j.amjoto.2020.10...

The incubation period in children is generally of two days, but can vary from 2 to 10 days.66. Dong Y, Mo X, Hu Y, Qi X, Jiang F, Jiang Z, et al. Epidemiology of COVID-19 Among Children in China. Pediatrics. 2020 Jun;145(6):145. https://doi.org/10.1542/peds.2020-0702
https://doi.org/10.1542/peds.2020-0702...
The clinical classification and characteristics of the novel coronavirus infections are described in Table 1. Although symptomatic cases usually attract more attention, asymptomatic children should be closed monitored as they can play a significant role in the transmission of SARS-CoV-2 infections.1515. Shen K, Yang Y, Wang T, Zhao D, Jiang Y, Jin R, et al. Diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts’ consensus statement. World J Pediatr. 2020 Jun;16(3):223-31. https://doi.org/10.1007/s12519-020-00343-7
https://doi.org/10.1007/s12519-020-00343...
Asymptomatic infected children are defined as those who do not have any clinical signs and symptoms or imaging findings of the disease such as the ground glass patching opacities in the thorax tomography, but tested positive for COVID-19 by RT-PCR or a serological method.33. Patel NA. Pediatric COVID-19: systematic review of the literature. Am J Otolaryngol. 2020 Sep - Oct;41(5):102573. https://doi.org/10.1016/j.amjoto.2020.102573
https://doi.org/10.1016/j.amjoto.2020.10...

Table 1
Clinical classifications of the characteristics of the 2019 novel coronavirus infections in children according to an expert consensus statement.1515. Shen K, Yang Y, Wang T, Zhao D, Jiang Y, Jin R, et al. Diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts’ consensus statement. World J Pediatr. 2020 Jun;16(3):223-31. https://doi.org/10.1007/s12519-020-00343-7
https://doi.org/10.1007/s12519-020-00343...

On the other hand, symptomatic infected children may have fever, dry cough, fatigue, upper airway respiratory symptoms (e.g. nasal congestion and runny nose) and gastrointestinal symptoms (e.g. abdominal discomfort, nausea, vomiting, abdominal pain and diarrhea).1515. Shen K, Yang Y, Wang T, Zhao D, Jiang Y, Jin R, et al. Diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts’ consensus statement. World J Pediatr. 2020 Jun;16(3):223-31. https://doi.org/10.1007/s12519-020-00343-7
https://doi.org/10.1007/s12519-020-00343...
,1616. Credie GB, Coelho AA, Rezende KM. Coronavirus (COVID-19) in children: history and pediatric oral health. Dental Oral Biol Craniofac Res. 2020; 3(3):1-4. https://doi.org/10.31487/j.DOBCR.2020.03.06
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Although the literature shows that there are several cases of mildly affected children, SARS-CoV-2 may cause a severe multi-systemic hyper-inflammatory reaction in genetically predisposed children, described as a Kawasaki's disease. This condition is likely to have a viral etiology and myocardial cell tropism, causing myocarditis and affecting the coronary arteries.1717. Loomba RS, Villarreal E, Flores S. COVID-19 and Kawasaki syndrome: should we really be surprised? Cardiol Young. 2020 Jul;30(7):1059-60. https://doi.org/10.1017/S1047951120001432
https://doi.org/10.1017/S104795112000143...
The diagnosis criteria for Kawasaki's disease is the presence of fever for five days with at least four out of five clinical criteria, in the absence of an alternative diagnosis.88. Jones VG, Mills M, Suarez D, Hogan CA, Yeh D, Segal JB, et al. COVID-19 and Kawasaki disease: novel virus and novel case. Hosp Pediatr. 2020 Jun;10(6):537-40. https://doi.org/10.1542/hpeds.2020-0123
https://doi.org/10.1542/hpeds.2020-0123...

Jones et al.,88. Jones VG, Mills M, Suarez D, Hogan CA, Yeh D, Segal JB, et al. COVID-19 and Kawasaki disease: novel virus and novel case. Hosp Pediatr. 2020 Jun;10(6):537-40. https://doi.org/10.1542/hpeds.2020-0123
https://doi.org/10.1542/hpeds.2020-0123...
were among the first authors who reported a case of a 6-month-old infant with COVID-19 infection and Kawasaki's disease. According to them, the child presented with fever, restlessness, refusal to eat, irritability, limbic sparing conjunctivitis, dry cracked lips, and the presence of an erythematous, apparently non-pruritic, blotchy cutaneous rash, mild sub-costal retractions suggestive of a respiratory distress and faint opacities were observed in the middle area of the left lung on the chest radiography. The echocardiogram was normal.

As far as we are concerned, there is not yet a guideline especially devoted to children with COVID-19 and comorbidities, such as chronic respiratory diseases, immune suppression conditions, uncorrected heart disease and chronic kidney disease. This population needs better surveillance, early diagnosis and supportive treatment.1414. Sankar J, Dhochak N, Kabra SK, Lodha R. COVID-19 in Children: Clinical Approach and Management. Indian J Pediatr. 2020 Jun;87(6):433-42. https://doi.org/10.1007/s12098-020-03292-1
https://doi.org/10.1007/s12098-020-03292...

Re-opening of schools and saliva as a diagnostic fluid

Due to the COVID-19 pandemic, kindergartens and schools were closed in an attempt to restrain the dissemination of the virus among children, the school staff, and their families. This measure has been discussed by UNESCO together with UNICEF and WHO. In general, the closure of schools causes a negative impact on child learning1818. Organização das Nações Unidas para a Educação,a Ciência e a Cultura – Unicef. Recomendações para a reabertura de escolas. Apr. 2020 [cited year Month day]. Available from: https://www.unicef.org/media/68886/file/PORTUGUESE-Framework-for-reopening-schools-2020.pdf
https://www.unicef.org/media/68886/file/...
besides puts additional economic and psychological stress on families.1919. Centers for Disease Control and Prevention – CDC. Preparing K-12 school administrators for a safe return to school in fall 2020. 2020 [cited year Month day]. Available from:

However, the re-opening of schools should only take place after ensuring that all students and school employees are safe,2020. Cooper DM, Guay-Woodford L, Blazar BR, Bowman S, Byington CL, Dome J, et al. Reopening Schools safely: the case for collaboration, constructive disruption of Pre-Coronavirus 2019 expectations, and creative solutions. J Pediatr. 2020 Aug;223:183-5. https://doi.org/10.1016/j.jpeds.2020.05.022
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which is consist with the sanitation measures taken by each country.1818. Organização das Nações Unidas para a Educação,a Ciência e a Cultura – Unicef. Recomendações para a reabertura de escolas. Apr. 2020 [cited year Month day]. Available from: https://www.unicef.org/media/68886/file/PORTUGUESE-Framework-for-reopening-schools-2020.pdf
https://www.unicef.org/media/68886/file/...
It is known that, in view of the current circumstances, children may be at real risk of contracting COVID-19 infection regardless of their age.88 According to Viner et al.,2121. Viner RM, Russell SJ, Croker H, Packer J, Ward J, Stansfield C, et al. School closure and management practices during coronavirus outbreaks including COVID-19: a rapid systematic review. Lancet Child Adolesc Health. 2020 May;4(5):397-404. https://doi.org/10.1016/S2352-4642(20)30095-X
https://doi.org/10.1016/S2352-4642(20)30...
children and adolescents aged between 10 and 19 years old can be as vulnerable to the new coronavirus infection as adults. Another study showed that the replication of SARS-CoV-2 in older children led the viral load to levels similar to those found in adults, whereas significantly higher amounts are detected in children younger than 5 years old.2020. Cooper DM, Guay-Woodford L, Blazar BR, Bowman S, Byington CL, Dome J, et al. Reopening Schools safely: the case for collaboration, constructive disruption of Pre-Coronavirus 2019 expectations, and creative solutions. J Pediatr. 2020 Aug;223:183-5. https://doi.org/10.1016/j.jpeds.2020.05.022
https://doi.org/10.1016/j.jpeds.2020.05....
Because all these age groups correspond to the school-age population, the return to school could lead to new outbreaks of the disease as children and adolescents may not respect the safety measures, thus increasing the risk of contamination.

There are some protocols being used in commercial places for restraining the dissemination of the virus, namely: cleaning the shoes before entering the building, measuring the body temperature, wearing face mask mandatorily, washing hands with soap or rubbing them with 70% alcohol, and keeping social distancing of about 1.5 meter from another person.1919. Centers for Disease Control and Prevention – CDC. Preparing K-12 school administrators for a safe return to school in fall 2020. 2020 [cited year Month day]. Available from:,2222. Walger P, Heininger U, Knuf M, Exner M, Popp W, Fischbach T, et al. Children and adolescents in the CoVid-19 pandemic: schools and daycare centers are to be opened again without restrictions. The protection of teachers, educators, carers and parents and the general hygiene rules do not conflict with this. GMS Hyg Infect Control. 2020 May;15: Doc11. https://doi.org/10.3205/dgkh000346
https://doi.org/10.3205/dgkh000346...

The document entitled “Considerations for school-related public health measures in the context of COVID-19”, developed with input from the Technical Advisory Group (TAG) of Experts on Educational Institutions and COVID-19 and experts from WHO, UNICEF, and UNESCO, reports some more specific indications for school operations in the context of COVID-19.2323. World Health Organization – WHO. Considerations for school-related public health measures in the context of COVID-19. Geneva: World Health Organization; 2020. Table 2 shows the summarised information of this document.2323. World Health Organization – WHO. Considerations for school-related public health measures in the context of COVID-19. Geneva: World Health Organization; 2020. This guidance is relevant for children, parents or caregivers, teachers and other staff, besides having an impact on the general society.

Table 2
Summary of considerations for school-related public health measures in context of COVID-19.2323. World Health Organization – WHO. Considerations for school-related public health measures in the context of COVID-19. Geneva: World Health Organization; 2020.

Thus, thinking about the function and importance of schools for the intellectual and social development of children, the re-opening must be done safely. For this reason, large-scale testing using saliva2424. Fernandes LL, Borges L, Pacheco VB, Bernardes A, Ribeiro RR, Jimenez M, et al. SARS-CoV-2: a promising path in salivary diagnosis. Open Dent J. 2020;14(1):2. https://doi.org/10.2174/1874210602014010343
https://doi.org/10.2174/1874210602014010...
can be a viable alternative to ensure testing of all children and school staff before and during the re-opening of the schools. Hence, depending on the child's age and level of understanding about the pandemic, complying with safety rules can be complicated. In addition, the suspicion of positive COVID-19 can be more difficult, especially when they are asymptomatic, making it difficult to screen for SARS-CoV-2.

Saliva-based diagnosis and screening of COVID-19 in children

Many studies report that blood has a direct relationship with saliva due to the existence of cellular and molecular bonds between these fluids.2525. Cross BW, Ruhl S. Glycan recognition at the saliva - oral microbiome interface. Cell Immunol. 2018 Nov;333:19-33. https://doi.org/10.1016/j.cellimm.2018.08.008
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It is suggested that both salivary IgG and IgM are derived from blood. Saliva is a mixture of secretions of different salivary glands, desquamated oral epithelia, gingival crevicular fluid and several microorganisms. It also contains a large number of proteins, such as immunoglobulins, in particular of secreted IgA, mucins, and enzymes, as well as metabolites, hormones, and electrolytes.2626. Ceron JJ, Lamy E, Martinez-Subiela S, Lopez-Jornet P, Capela ESF, Eckersall PD, Tvarijonaviciute A. Use of saliva for diagnosis and monitoring the SARS-CoV-2: a general perspective. J Clin Med. 2020 May;9(5):1491. https://doi.org/10.3390/jcm9051491
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This composition allows the detection of pathogens in saliva and also the quantification of biomarkers that can provide information on the immunological, inflammatory, endocrine, and metabolic status of each individual.2727. Javaid MA, Ahmed AS, Durand R, Tran SD. Saliva as a diagnostic tool for oral and systemic diseases. J Oral Biol Craniofac Res. 2016 Jan-Apr;6(1):66-75. https://doi.org/10.1016/j.jobcr.2015.08.006
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,2828. Pedersen AM, Sørensen CE, Proctor GB, Carpenter GH, Ekström J. Salivary secretion in health and disease. J Oral Rehabil. 2018 Sep;45(9):730-46. https://doi.org/10.1111/joor.12664
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,2929. Zhang CZ, Cheng XQ, Li JY, Zhang P, Yi P, Xu X, et al. Saliva in the diagnosis of diseases. Int J Oral Sci. 2016 Sep;8(3):133-7. https://doi.org/10.1038/ijos.2016.38
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,3030. Marques Filho JS, Gobara Junior J, Salomao GVS, Sumita LM, Shibli JA, Viana RG, et al. cytokine levels and human herpesviruses in saliva from clinical periodontal healthy subjects with peri-implantitis: a case-control study. Mediators Inflamm. 2018 Aug;2018:6020625. https://doi.org/10.1155/2018/6020625
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Viral infections can be diagnosed in the saliva by directly targeting the genetic material of the virus (i.e. DNA or RNA) or indirectly, by searching for signatures left by these infections, such as proteins (proteomic analysis), metabolites (metabolomic analysis), miRNAs and other transcriptomic analyses and immunoglobulin profiles (IgA, IgG and IgM).3131. To KK, Chan KH, Ho J, Pang PK, Ho DT, Chang AC, et al. Respiratory virus infection among hospitalized adult patients with or without clinically apparent respiratory infection: a prospective cohort study. Clin Microbiol Infect. 2019 Dec;25(12):1539-45. https://doi.org/10.1016/j.cmi.2019.04.012
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Immunoglobulins such as IgM, IgG and IgA, the latter abundant in saliva, can act as biomarkers in the saliva, allowing this fluid to be used for diagnosing and monitoring of these infections.3232. Castro T, Sabalza M, Barber C, Abrams W, Costa AC, Milagres FAP, et al. Rapid diagnosis of Zika virus through saliva and urine by Loop-mediated isothermal amplification (LAMP). J Oral Microbiol. 2018 Sep;10(1):1510712. https://doi.org/10.1080/20002297.2018.1510712
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In addition, they also enable the development of point-of-care diagnostic devices, which are extremely useful for mass application.3333. Braz-Silva PH, Pallos D, Giannecchini S, To KK. SARS-CoV-2: what can saliva tell us? Oral Dis. 2020 Apr;odi.13365. https://doi.org/10.1111/odi.13365
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SARS-CoV-2 has been found in saliva on a sustained basis.3434. To KK, Tsang OT, Yip CC, Chan KH, Wu TC, Chan JM, et al. Consistent detection of 2019 novel coronavirus in saliva. Clin Infect Dis. 2020 Jul;71(15):841-3. https://doi.org/10.1093/cid/ciaa149
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The viral particles found in the oral cavity can come from different sources, such as the upper respiratory tract, crevicular gingival fluid and/or come directly from the salivary glands.3535. To KK, Tsang OT, Leung WS, Tam AR, Wu TC, Lung DC, et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis. 2020 May;20(5):565-74. https://doi.org/10.1016/S1473-3099(20)30196-1
https://doi.org/10.1016/S1473-3099(20)30...

In fact, asymptomatic individuals do not present with a typical respiratory pattern despite the presence of viable virus particles in their oral fluids.3636. Long QX, Tang XJ, Shi QL, Li Q, Deng HJ, Yuan J, et al. Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections. Nat Med. 2020 Aug;26(8):1200-4. https://doi.org/10.1038/s41591-020-0965-6
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In this sense, in the context of other infectious diseases, some studies have shown that it is possible to detect the presence of different viruses in saliva samples.3535. To KK, Tsang OT, Leung WS, Tam AR, Wu TC, Lung DC, et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis. 2020 May;20(5):565-74. https://doi.org/10.1016/S1473-3099(20)30196-1
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,3737. Barzon L, Pacenti M, Berto A, Sinigaglia A, Franchin E, Lavezzo E, et al. Isolation of infectious Zika virus from saliva and prolonged viral RNA shedding in a traveller returning from the Dominican Republic to Italy, January 2016. Euro Surveill. 2016;21(10):30159. https://doi.org/10.2807/1560-7917.ES.2016.21.10.30159
https://doi.org/10.2807/1560-7917.ES.201...
,3838. Zuanazzi D, Arts EJ, Jorge PK, Mulyar Y, Gibson R, Xiao Y, et al. Postnatal Identification of Zika Virus Peptides from Saliva. J Dent Res. 2017 Sep;96(10):1078-84. https://doi.org/10.1177/0022034517723325
https://doi.org/10.1177/0022034517723325...

Saliva collection is a simpler and cheaper procedure, in addition to being better tolerated by children and their parents, which makes this method even more attractive for the pediatrician as it is a minimally invasive approach.3939. Azzi L, Carcano G, Gianfagna F, Grossi P, Gasperina DD, Genoni A, et al. Saliva is a reliable tool to detect SARS-CoV-2. J Infect. 2020 Jul;81(1):e45-50. https://doi.org/10.1016/j.jinf.2020.04.005
https://doi.org/10.1016/j.jinf.2020.04.0...
Another advantage of this collection method is that it can be safely performed by the children's parents, which significantly reduces the costs of COVID-19 testing, as no specialised professional is needed for the sample collection, and consequently, there is no risk of exposure for the healthcare team.4040. Sabino-Silva R, Jardim AC, Siqueira WL. Coronavirus COVID-19 impacts to dentistry and potential salivary diagnosis. Clin Oral Investig. 2020 Apr;24(4):1619-21. https://doi.org/10.1007/s00784-020-03248-x
https://doi.org/10.1007/s00784-020-03248...

Saliva collection techniques for children

There is a great variety of saliva collection methods, such as aspiration with syringe and test tube, open or closed suction, expectoration or spit, drainage and swab. The choice of the method for children should be made according to their age and degree of understanding (Table 3).4141. Klimes-Dougan B, Hastings PD, Granger DA, Usher BA, Zahn-Waxler C. Adrenocortical activity in at-risk and normally developing adolescents: individual differences in salivary cortisol basal levels, diurnal variation, and responses to social challenges. Dev Psychopathol. 2001;13(3):695-719. https://doi.org/10.1017/S0954579401003157
https://doi.org/10.1017/S095457940100315...

Table 3
Summary of the saliva collection techniques according to the children's age group.

To perform the salivary tests, it is not necessary to perform tooth brushing or make a mouthwash.4242. Navazesh M. Saliva in health and disease. J Calif Dent Assoc. 2011 Sep;39(9):626-8. As the diagnosis is also for virus, this type of test can be made at any moment of the day. An important aspect to be considered is that the amount of saliva to be collected should be around 3 mL, which can be accomplished with or without stimulation. In case of saliva production after stimulation, the collection procedure can be performed mechanically (e.g. chewing gum, paraffin, latex) or chemically (e.g. citric acid at 1–5%).4343. Hanrahan K, McCarthy AM, Kleiber C, Lutgendorf S, Tsalikian E. Strategies for salivary cortisol collection and analysis in research with children. Appl Nurs Res. 2006 May;19(2):95-101. https://doi.org/10.1016/j.apnr.2006.02.001
https://doi.org/10.1016/j.apnr.2006.02.0...
Irrespective of the collection method chosen, it is a challenge to collect enough saliva from neonates and infants up to 3 months of life, as this group of children in general produce very little saliva.4444. Herrington CJ, Olomu IN, Geller SM. Salivary cortisol as indicators of pain in preterm infants: a pilot study. Clin Nurs Res. 2004 Feb;13(1):53-68. https://doi.org/10.1177/1054773803259665
https://doi.org/10.1177/1054773803259665...
In the literature, it is recommended to drip some drops of 5% citric acid onto the sub-lingual floor in order to stimulate an increased production of saliva,4545. Walker RF, Riad-Fahmy D, Read GF. Adrenal status assessed by direct radioimmunoassay of cortisol in whole saliva or parotid saliva. Clin Chem. 1978 Sep;24(9):1460-3. https://doi.org/10.1093/clinchem/24.9.1460
https://doi.org/10.1093/clinchem/24.9.14...
which will therefore be aspirated with syringe and test tube.4646. Klug I, Dressendörfer R, Strasburger C, Kühl GP, Reiter HL, Reich A, et al. Cortisol and 17-hydroxyprogesterone levels in saliva of healthy neonates: normative data and relation to body mass index, arterial cord blood ph and time of sampling after birth. Biol Neonate. 2000 Jul;78(1):22-6. https://doi.org/10.1159/000014242
https://doi.org/10.1159/000014242...

In children older than 6 months, the salivary glands in the oral cavity region begin to produce saliva spontaneously and regularly, which certainly make its collection easier. This can be carried out in two ways, namely, by aspirating the saliva with syringe and test tube or by using the open suction technique.4747. Jones JM, Watkins CA, Hand JS, Warren JJ, Cowen HJ. Comparison of three salivary flow rate assessment methods in an elderly population. Community Dent Oral Epidemiol. 2000 Jun;28(3):177-84. https://doi.org/10.1034/j.1600-0528.2000.280303.x
https://doi.org/10.1034/j.1600-0528.2000...

In the open suction technique, the saliva is aspirated through a plastic tube, which is connected to a portable suction machine, into a container where it will be collected and stored. For this type of collection, the child should be encouraged to keep the mouth open for 15 seconds, on average, at intervals of two minutes.4747. Jones JM, Watkins CA, Hand JS, Warren JJ, Cowen HJ. Comparison of three salivary flow rate assessment methods in an elderly population. Community Dent Oral Epidemiol. 2000 Jun;28(3):177-84. https://doi.org/10.1034/j.1600-0528.2000.280303.x
https://doi.org/10.1034/j.1600-0528.2000...
There is also the closed suction technique, in which a saliva collection tube is placed below the tongue and the child is asked to keep the lips closed around it for two minutes.4747. Jones JM, Watkins CA, Hand JS, Warren JJ, Cowen HJ. Comparison of three salivary flow rate assessment methods in an elderly population. Community Dent Oral Epidemiol. 2000 Jun;28(3):177-84. https://doi.org/10.1034/j.1600-0528.2000.280303.x
https://doi.org/10.1034/j.1600-0528.2000...
However, this technique is indicated only for children older than 3 years.

For children older than 6 years, saliva can be collected by using the expectoration or spit method, in which the child should keep the mouth closed for three minutes without swallowing so that the saliva is accumulated and then spit into the collection tube.4747. Jones JM, Watkins CA, Hand JS, Warren JJ, Cowen HJ. Comparison of three salivary flow rate assessment methods in an elderly population. Community Dent Oral Epidemiol. 2000 Jun;28(3):177-84. https://doi.org/10.1034/j.1600-0528.2000.280303.x
https://doi.org/10.1034/j.1600-0528.2000...
However, because this technique may be difficult to perform in some children due to their difficulty in not swallowing the saliva for a given period of time, the drainage technique can be a viable alternative. In the drainage technique, the child is initially asked to swallow the saliva, so that new amounts of saliva are produced before being drained through the half-opened lips and directly stored into the collection tube.

The swab method can also be used as an alternative for collecting saliva. For doing so, three cotton rolls are placed in different sites of the oral cavity: one below the tongue in order to absorb the saliva close to the excretory duct orifices of the sub-mandibular and sub-lingual glands, and the other two cotton rolls close to the excretory ducts orifices of the parotid glands, one on each side of the oral cavity. The cotton rolls should remain inside the child's oral cavity for 2-3 minutes, on average, so that they are soaked with saliva. At the end of the saliva collection, the cotton rolls are removed and placed into a test tube before being properly closed. The recovery of the absorbed saliva occurs by means of centrifugation of the test tube containing the cotton rolls inside,4848. Strazdins L, Meyerkort S, Brent V, D’Souza RM, Broom DH, Kyd JM. Impact of saliva collection methods on sIgA and cortisol assays and acceptability to participants. J Immunol Methods. 2005 Dec;307(1-2):167-71. https://doi.org/10.1016/j.jim.2005.09.010
https://doi.org/10.1016/j.jim.2005.09.01...
which results in a sample of clear fluid. This technique requires no cold storage, a condition that is frequently used in other methods for saliva collection.4848. Strazdins L, Meyerkort S, Brent V, D’Souza RM, Broom DH, Kyd JM. Impact of saliva collection methods on sIgA and cortisol assays and acceptability to participants. J Immunol Methods. 2005 Dec;307(1-2):167-71. https://doi.org/10.1016/j.jim.2005.09.010
https://doi.org/10.1016/j.jim.2005.09.01...
,4949. Navazesh M, Mulligan RA, Kipnis V, Denny PA, Denny PC. Comparison of whole saliva flow rates and mucin concentrations in healthy Caucasian young and aged adults. J Dent Res. 1992 Jun;71(6):1275-8. https://doi.org/10.1177/00220345920710060201
https://doi.org/10.1177/0022034592071006...
,5050. Sapkota D, Søland TM, Galtung HK, Sand LP, Giannecchini S, To KKW, et al. COVID-19 salivary signature: diagnostic and research opportunities. J Clin Pathol. 2020;0:1-6. https://doi.org/10.1136/jclinpath-2020-206834
https://doi.org/10.1136/jclinpath-2020-2...

Conclusion

COVID-19 symptomatic and asymptomatic patients can be diagnosed by means of saliva, whose advantage is to be less invasive, simpler, cheaper and relatively quickly sampled. In addition, the risk of dissemination for healthcare professional is reduced as the saliva can be collected by the own individual or the children's parents. Another important advantage of saliva is that it can be serially collected, which is a key factor in a dynamic infection context, as is the case of COVID-19. Another point to be highlighted is that saliva is an excellent fluid to detect not only the virus genetic material, but also signatures of previous infections by using proteins (proteomic), metabolites (metabolomic), micro RNAs and transcriptomic and immunoglobulins, mainly IgA analyses.

In view of this, saliva can be considered an alternative biological fluid for screening and diagnosing COVID-19 in children, thus playing a key role in the understanding of the behaviour of the infection in childhood.

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    Strazdins L, Meyerkort S, Brent V, D’Souza RM, Broom DH, Kyd JM. Impact of saliva collection methods on sIgA and cortisol assays and acceptability to participants. J Immunol Methods. 2005 Dec;307(1-2):167-71. https://doi.org/10.1016/j.jim.2005.09.010
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Publication Dates

  • Publication in this collection
    20 Nov 2020
  • Date of issue
    2020

History

  • Received
    09 Sept 2020
  • Reviewed
    29 Oct 2020
  • Accepted
    30 Oct 2020
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