FormalPara Key Points

This scoping review contributes to the body of evidence, as it give clinicians valuable information while choosing a saliva collecting method that is best suited for use in children and adolescents.

For uncooperative children and those under the age of 6 years, the suction and absorption methods of saliva collection are the most preferred. In adolescents and older children, no specific method was found to be preferred over another method.

1 Introduction

Saliva is a clear, sero-mucousus exocrine secretion. This biological fluid contains a wide variety of biomolecules that present several functions. For example, saliva plays a role in wound healing and blood clotting, has a buffering capacity, protects oral tissues against infection and inflammation, and prevents dehydration of the hard and soft tissues [1]. Therefore, this secretion has a very important role in preserving and maintaining oral health, and plays a major role in maintaining an individual’s quality of life [2].

Saliva is produced by three pairs of major glands and numerous minor salivary glands. The three major salivary glands include the paired parotid, submandibular, and sublingual glands. On average, the major glands are responsible for 90% of the total saliva secretion and the minor glands contribute approximately 10% of its volume during the day [3]. Each gland secretes a unique palette of compounds into the oral cavity. For example, the parotid gland secretes alfa-amylase rich saliva. In contrast, the submandibular and sublingual glands secrete saliva enriched with the glycoprotein MUC5B [4]. Thus, the salivary glands secrete types of compounds that are differentially expressed in specific secretions of the glands.

Next, dynamics in salivary composition is sensitive to stimuli. For instance, unstimulated saliva is about two-thirds produced by the submandibular glands, while under chew or taste stimulation, the parotid glands can increase secretion to a value up to 70% of the total volume. At either rest or stimulation, the sublingual glands contribute with a minor percentage. Thus, by examining the composition of saliva, the site of production and contribution of each gland can be defined [4, 5]. In addition, multiple diseases and salivary gland disorders can be screened or diagnosed by saliva collection and subsequent analysis. Some inflammatory diseases such as gingivitis and periodontitis, but also Sjögren’s disease, are examples of which can be identified through analyzing saliva [6, 7], supporting routine diagnostics and choice of therapy [2, 8].

Compared to other diagnostic matrices such as blood or urine, saliva collection is associated with fewer compliance problems and complications and is, therefore, of value to both children and adults. The main advantages are that it is a simple, cost-effective, and non-invasive screening and/or diagnostic approach. Furthermore, it is painless and does not require any sterile equipment or medically trained personnel [9]. However, it is crucial to use standardized methods for saliva collection to ensure the reliability and reproducibility of clinical and research findings. According to Shannon and Chauncey [10], the most optimal way for saliva collection, for both stimulated and unstimulated saliva, is with the eyes open, sitting up straight, with the head tilted a bit forward. Unfortunately, not all patient groups can simply adopt this stance. Young children, for instance, may experience more difficulty in this regard; therefore, collecting saliva, which is considered a simple procedure, can be a challenge in this patient group. Furthermore, the method of saliva collection is related to the type of saliva to be analyzed [11]. There are multiple ways to collect saliva, with the most prevalent methods being the passive drooling method, spitting method, absorption method, and suction method. The drooling method is commonly used to collect the unstimulated saliva, while the spitting, suction, and absorption methods are utilized to sample the stimulated saliva [12], and this stimulation can be achieved through chewing and gustatory stimulation (e.g., citric acid) [13].

Due to factors such as willingness to cooperate, age and development stage of the child, unfamiliarity with the collection process, and feelings of safety, it seems plausible that certain collection methods are preferred over others when applied in children [14]. In particular, it is important to ensure that the child is comfortable and relaxed during the collection procedure to avoid an unpleasant experience. Hence, in this scoping review, we aimed to comprehensively appraise the different methods of saliva collection among children and adolescents. In addition, we investigated which methods are preferred by patients and clinicians. The outcome of this study can assist in identifying a safe and most effective way to collect saliva among children and adolescents.

2 Materials and Methods

2.1 Search Strategy

This scoping review was conducted adhering to the methodological framework of Arksey and O’Malley [15], and was reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews (PRISMA-ScR) checklist [16]. A systematic search was performed in the bibliographic databases of the National Library of Medicine (PubMed-MEDLINE), Embase and Web of Science. The search strategy included search terms such as general controlled vocabulary (Medical Subject Headings (MeSH) in PubMed and Emtree in Embase), synonymous, related terms, or free text words. The following terms were used: ‘saliva’ and ‘specimen handling’ and ‘child’ or ‘adolescent’. The search was adapted according to the different databases. The specific search details for each database are provided in Table 1. No restriction was applied regarding the date of publication. Additional studies were identified by verifying the reference lists of relevant articles.

Table 1 Search strategy of the various databases
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2.2 Inclusion and Exclusion Criteria

Inclusion criteria for this review were: (1) studies describing the saliva collection or sampling process with the collection method or sampling device used; (2) studies carried out in patients aged from 0 to 18 years; (3) studies carried out in patients with good overall health and those with only mild systemic conditions that did not significantly impact their functional abilities or studies with a healthy control group; (4) cohort studies, cross-sectional studies, case–control studies, and randomized or quasi-randomized clinical trials; and (5) articles published in English. The predefined exclusion criteria were: (1) articles where full text was not available or full text could not be retrieved; (2) literature reviews, editorials, case reports, conference papers, and systematic reviews; (3) studies that do not yield original data; (4) studies including participants with orthodontic appliances or splints; (5) studies that did not report data of adults separated from children or adolescents on the outcome measurement; (6) patients using drugs, medicines, or mouthwashes, except for studies where saliva was collected as a baseline before starting any (medical) treatment (in those cases we only considered the data collected at baseline); (7) artificial saliva. Also, studies carried out in (8) participants who had chronic diseases or conditions that could affect salivary function, such as Sjögren’s disease and rheumatoid arthritis, or without a healthy control group were excluded [17].

2.3 Screening Process and Data Selection

The process of study selection is described in Fig. 1. Obtained studies from the search strategy were entered into EndNote 20 and RAYYAN, an online Web application for screening literature reviews. Duplicates were excluded electronically in EndNote. Evaluation of the potentially relevant titles and abstracts was performed independently by two reviewers (JMHF and DH). Prior to the screening of the titles and abstracts, calibration was conducted between the two reviewers. For this, 50 randomly selected articles were screened independently by the two reviewers (kappa = 0.95). The studies that complied with the inclusion criteria were retained for full-text review, which was performed by one reviewer (JMHF). The full-text articles were checked for eligibility and the second reviewer (DH) controlled the outcome of the selection for its comprehensiveness and accuracy. Inconsistencies or doubts were resolved through discussion and consensus between the two reviewers. If no agreement was reached, a third reviewer (FB) was consulted. The reference lists of the selected articles were screened for potentially relevant studies, which were not found in the search. Publications that fulfilled all selection criteria were incorporated for data extraction.

Fig. 1
figure 1

PRISMA flowchart of the literature search and data selection

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2.4 Data Extraction and Summary

One reviewer (JMHF) extracted the data from the studies included. Information on articles, such as: author, year of publication, country of origin of article, study design, sample size, age range of the participants, the aim of the study, method of saliva collection, remarks on sampling (if applicable), the type of saliva that was collected, and main outcome of the study were collected and recorded in an Excel spreadsheet. The primary interest was to comprehensively appraise the different methods of saliva collection. The second interest was to determine which methods of saliva collection were most suitable for children or adolescents. A method was considered suitable if (1) the right type and proper quantity of saliva could be collected, (2) the patient did not find it uncomfortable and it was preferred for their age or degree of understanding, and if (3) the child's treatability was good. One reviewer (JMHF) collected all the relevant data, and a second reviewer (DH) verified its completeness and accuracy. The extracted data were summarized in a narrative synthesis.

3 Results

3.1 Search Results

The systematic literature search generated a total of 2569 studies. After removing duplicates, 1853 studies were identified, and screening of titles and abstracts was conducted by the two reviewers. A total of 562 studies remained for a detailed full-text analysis. Following the study selection procedure, a total of 249 studies were ultimately eligible for inclusion for this scoping review. No studies were added after reviewing the reference list of the included articles. All studies that were selected included a detailed description of various methods for collecting saliva in children and adolescents (\(\le\) 18 years), accompanied by an elucidation of the specific sampling device employed in each instance. The PRISMA flowchart depicting the literature search and selection procedure, as well as the reasons for exclusion, is provided in Fig. 1.

3.2 Study Characteristics

The descriptive characteristics of the included studies are given in the Online Supplementary Material (OSM; Table S1). Among the included studies, 13 were case–control studies, 12 were cohort studies, 205 were cross-sectional studies, 17 were randomized controlled trials (RCTs), and two were non-randomized controlled trials (NRCTs). These studies were published between 1987 and 2022, originating from 48 countries. The USA was represented the most in the articles (n = 38). A total of 89,368 saliva samples have been collected from all the participants. The most commonly used collection method was the absorption method (n = 113), followed by the spitting method (n = 85), the drooling method (n = 42), and finally the suction method (n = 34). Although most studies implemented only one saliva collection method (n = 230), some authors combined the use of two (n = 16) or three (n = 3) different methods. In 114 studies, patients were asked to fast before saliva collection (no eating, drinking, or brushing their teeth). The duration of fasting could vary from 10 min [18] to an entire evening before sampling [19,20,21,22,23,24,25,26,27,28]. In addition to the different collection methods, different types of saliva were also collected. Specifically, unstimulated saliva (n = 207), stimulated saliva (n = 48), and unstimulated (n = 1) or stimulated parotid saliva (n = 2) were collected. Furthermore, several studies obtained not only one but multiple types of saliva (n = 7) from the participants.

The distribution of studies according to the method used for saliva collection and the types of saliva collected, namely unstimulated (US) and stimulated (SS), are illustrated in an alluvial diagram in Fig. 2. The diagram illustrates the different methods using color coding, where red represents suction, orange represents drooling, green represents spitting, and blue represents absorption. The height of the flow fields demonstrates the number of studies that connected the various types of saliva through the flow fields, whereas the height of each block refers to the number of studies that used a specific collection method. It can be seen that the absorption method was the method most often used, and all four saliva collection methods are used mainly for collecting unstimulated saliva.

Fig. 2
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Alluvial diagram illustrating the saliva collection methods and corresponding salivary types. SS stimulated saliva, US unstimulated saliva

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3.3 Saliva Collection Methods

3.3.1 Drooling Method

A total of 42 studies employed the drooling method to collect the saliva from both children and adolescents. While 33 studies solely relied on drooling for the saliva collection, nine other studies also utilized additional collection methods. In most of these studies (n = 39), unstimulated saliva was collected, with the exception of Whitford et al. [29], where stimulated parotid saliva was sampled, as well as in the studies of Granger et al. [30] and Carvalho and Bezerra [31], where stimulated saliva was collected. The drooling method was often preferred because of its high acceptance rate for unstimulated saliva collection, and was considered as a simple and reproducible method [32]. In certain studies, the patients were instructed to rinse with water to eliminate any food debris and swallow the remaining saliva prior to the sampling [33,34,35]. Patients were asked to remain silent, to sit upright on the edge of their chair, keep their mouths open, and tilt their head slightly downward so that saliva could passively drain from the oral cavity and lower lip into a sterile collection device [36]. This position is also known as the “Coachman’s position” [37]. The duration of the collection and the amount of saliva required varied from study to study. The draining saliva was collected in a collection device, commonly a sterile tube, vial, or container. Occasionally, a straw, funnel, or chin cup could be employed to facilitate the process and minimize potential wastage [30, 38]. The SalivaBio Passive Drool Saliva Collection Aid (SCA) from Salimetrics served as the collection device in the studies conducted by Colceriu-Simon et al. [39], Westwell-Roper et al. [23], and Siani-Rose et al. [40]. In the study Dimitrijevic-Carlsson et al. [41], a modified Carlson-Crittenden collector was used to collect the unstimulated parotid saliva. After locating the Stensen's duct, gauze was used to dry the buccal mucosa in the area. The collector was positioned over Stensen's duct's papilla. A Lashley-cup was used to collect stimulated parotid saliva in the study by Whitford et al. [29]. During the saliva sampling, it is essential not to move the collection device as this disrupts the collection process. The patient must remain seated and sit still, and it is crucial to allocate a sufficient amount of time for the collection of saliva. In the study by Dimitrijevic-Carlsson et al. [41], cartoons were used to distract the children during the sampling. For adolescents and children aged 6 years and older this collection method worked well in terms of saliva collection effectiveness and acceptability of the patient. However, this was different for young children and infants. All studies using drooling methods were carried out in children older than 3 years of age or adolescents. Conversely, different saliva collecting methods were used while studying children under the age of 3 [42] or 5 [43] years in those studies. The drooling method was thus not applicable in each age group.

3.3.2 Absorption Method

The absorption method was the most commonly used saliva collection method in the included articles (n = 122). Both stimulated and unstimulated saliva could be sampled by this method. This method is often favored because of the ease of collection, the safety it offers, and the suitability for (young) children [20, 44]. The procedure for this collection method involves placing an absorbent device in the participant’s oral cavity for a specific period of time. The absorbent device subsequently absorbs the saliva, and a syringe, for instance, is utilized to express the saliva into a different collection device [45]. In many studies, a cotton roll, swab, or sponge was used to absorb the saliva. Examples of brands that produce these swabs, which were utilized in the included studies, include Sarstedt and Salimetrics. The Salivette from Sarstedt was most commonly used as the absorbent swab (n = 38). Salimetrics differentiates between regular swabs and swabs specifically designed for saliva collection in children under the age of 6 years. The SalivaBio Children’s Swabs (SCS) can be used for children from 6 years to 6 months and SalivaBio Infant’s Swab (SIS) for infants less than 6 months of age. The SIS was used in one included study [46] and the SCS in six other studies [47,48,49,50,51,52]. According to the available literature, there were no differences in the use of the absorption method between younger children and adolescents regarding the effectiveness of saliva collection, the quantity of saliva obtained, or the overall acceptability of the method. So, this method could be effectively applied across all age groups. The absorption method was also employed in situations whenever saliva collection using other methods such as spitting was not feasible in the patient group [53], or when children were uncooperative during the collection process [8], or when they were too young for other saliva collection techniques [44, 54].

3.3.3 Spitting Method

In 83 of the included studies the spitting method was utilized to collect the saliva among children and adolescents. Out of these, 12 studies used other saliva collection methods in addition to the spitting method. The spitting method operates in the following manner: The child is asked to gently expectorate the saliva in a sterile collection device for a duration of time [55]. The Coachman’s position, as previously described, is frequently utilized in this method as well [56]. In several studies the participants were asked to rinse their mouth with distilled or clean water a few seconds before the saliva collection [57, 58]. The spitting method allowed the collection of both stimulated and unstimulated saliva. The most commonly employed collection devices were disposable tubes, containers, cups, and vials. This sampling method is commonly preferred due to its high reproducibility and reliability in measuring the saliva flow rate [59]. However, the spitting method is not suitable for everyone. Some studies have reported difficulties that young children experienced when they tried to spit during the collection process, whether because they had a hard time following the researcher's instructions or as a result of their age-related restrictions. In adolescents and children older than 3 years of age, on the other hand, this method of saliva collection often provides reliable, reproducible, and successful results [60]. Therefore, this implies that this method could be applied in adolescents and older children but not in children under three years of age. In cases where it was not feasible for children to evacuate their saliva or if children were uncooperative, an alternative collection method was employed [54, 61, 62].

3.3.4 Suction Method

In total, 34 of the included studies utilized the suction method and nine of them combined it with other saliva collection methods. Both stimulated and unstimulated saliva could be collected using the suction method. No differences were mentioned in the literature in the use of the suction method between adolescents and younger children regarding the efficiency of saliva collection, the volume of saliva collected, or the general acceptability of the method. So, the suction method could be used in each age group. However, the suction method was frequently favored when obtaining saliva samples from infants, younger children or children with special needs was required [42, 61, 63]. The suction method operates in the following manner: Saliva accumulates in the bottom of the oral cavity and then, with gentle suction and low pressure, the saliva is aspirated through the created vacuum using a sterile collection device [64]. The most common collection devices were needleless syringes, (micro)pipettes, plastic catheters, or plastic droppers. Saliva yield by collection can be low in infants due to behavior-related challenges. However, the suction method using a micropipette or small needleless syringe still allowed an adequate amount of saliva to be collected [65]. Costa et al. [66] studied a newly developed portable device for saliva collection, the Sialometer. The exterior of the device was fitted with a monkey cover, specifically designed to hide all internal components (except for the timer display), and gave the appearance of a toy. A small toy monkey was integrated into the device and given to the child during saliva collection to assist with hand control. When the Sialometer was turned on, a vacuum was created inside the tube, which caused the saliva to be suctioned by a pressure differential. Even among children with more challenging conditions, the device had a great acceptability, which may have been made possible by the external characterization.

4 Discussion

The main aim of this research was to generate a comprehensive literature review on saliva collection methods among children and adolescents. Furthermore, we aimed to investigate which collection methods were preferred by the clinicians and patients in order to determine the most suitable and comfortable collection method for each age group or individuals with specific behavioral characteristics. Four distinct methods of saliva collection emerged from the included studies, namely: the drooling method, which involves the natural collection of saliva through drooling; the absorption method, in which saliva is collected using absorbent materials; the spitting method, in which patients actively expel saliva into a collection container; and the suction method, which employs suction devices to gather saliva from the patient.

Most studies used a single saliva collection method (n = 230); however, a subset of studies (n = 19) employed a combination of collection methods for various purposes. The main reasons for combining different methods included comparing different saliva collection methods within the same population [66,67,68,69], collecting different types of saliva or measurements [29, 38, 51, 70], aligning the method to the age or developmental stage of the participants [42, 43, 54, 61], in cases where destructive behavior posed challenges to the conventional approach [8, 64], or the interchangeability of different methods in certain instances [71,72,73].

Upon analyzing the four methods of saliva collection, it became evident that they varied considerably from one to another. Of the 249 studies included in this review, most of them (n = 122) used the absorption method for the saliva sampling, often employing a swab as the collection device. For instance, the brand Salimetrics has produced specialized swabs for two distinct age groups: children under 6 years (SCS) and infants under 6 months (SIS) of age. These swabs are designed with specific features to enhance their suitability for use with children, ensuring safety and comfort. Both types of swabs have a sufficient length, allowing for a secure grip with one hand while inserting the other end into the child's mouth, eliminating the risk of any choking hazard. Additionally, the swabs feature a thin diameter, well suited for use in smaller mouths, ensuring comfort and efficiency in the saliva collection process. The SIS swab, in particular, boasts an even slimmer diameter compared to the SCS swab (6.3 vs. 8 mm) [74, 75].

A remarkable finding is that across all studies that used the drooling method, the children were older than 3 years of age. On the other hand, studies that included children younger than 3 [42] or 5 [43] years of age predominantly utilized the absorption or suction method for saliva collection. This indicates a deliberate adaptation of the collection methods based on the patient's age. The drooling method, by its nature, requires a patient who is competent, compliant, aware, awake, and capable. While this method has proven effective for children over 3 years of age, as well as in the case of most adolescents and adults, it demonstrates limited efficacy when dealing with very young children under special circumstances. In such scenarios, the absorption or suction methods are preferred for saliva sampling [76]. In the absorption and suction methods, on the other hand, no differences were found in the use between children and adolescents regarding the effectiveness of saliva collection, the quantity of saliva obtained, or the overall acceptability of the method. This suggests that both methods are applicable to any age group.

The two main concerns for saliva collection in young children are safety and feasibility. While adolescents and older children often face no issues with saliva collection through methods like spitting or drooling, younger children frequently encounter difficulties due to their inability to spit, communication challenges, or maintaining the entire collection process. Consequently, these methods are generally applicable for adolescents and older children but prove less suitable for younger ones. In such situations, the use of a cotton swab or a needleless suction device has proven to be a straightforward and safe alternative, particularly well suited for younger children and infants [20, 62]. The SCA from Salimetrics was used as the collection device in the studies conducted by Colceriu-Simon et al. [39], Westwell-Roper et al. [23], and Siani-Rose et al. [40], all of which employed the drooling method for saliva collection. The SCA is purposefully designed to enhance the volume of saliva collected and promote participant compliance, making it a valuable tool in these research endeavors. However, it is imperative to emphasize that the use of the SCA is not advisable for children under 4 years of age, highlighting the necessity of age-appropriate selection of collection methods to ensure both effectiveness and safety in research and clinical practice.

An innovative collecting device, Salivac®, which is ingeniously designed as a pacifier, was introduced by Novak [77]. This unique device, resembling a regular pacifier, collects saliva as a child or newborn sucks on it. In comparison to the SCS from Salimetrics, the Salivac® demonstrated the ability to collect an equivalent volume of saliva while significantly reducing the time required by healthcare professionals for the collection process. It is important to note that, at present, this study represents the sole investigation into the use of the Salivac® saliva-collecting device. Another pioneering saliva collection device is the SaliPac®, which comprises a swab encased within an infant pacifier [78]. The SaliPac® successfully collects ample salivary volumes under 4 min, is user-friendly, and is well received by both children and caregivers. Despite the promising findings observed in these studies, it is essential to emphasize the need for further research in the future to draw definite and comprehensive conclusions regarding the effectiveness and practicality of these innovative saliva collection devices in both healthy and medically compromised populations.

Overall, the results of this scoping review indicate a generally high acceptance for the difference saliva collection methods among children and adolescents. All collection methods are considered to be simple, reliable, reproducible, and safe. However, it is important to note that not every collection method seems suitable for every age group. The key to a successful collection procedure lies in the appropriate method selection based on the age and developmental stage of the individual. Based on the present findings, when comparing these distinct collection methods, the suction or absorption methods emerge as the most favored options for infants or children under the age of 6 years. In contrast, the drooling and spitting methods are typically reserved for children aged 3 years and over due to the inherent challenges and duration associated with these collection processes. In situations where children exhibit unwillingness to cooperate during the collection procedure, the absorption method is often the most feasible choice. Remarkably, in the case of adolescents and older children, there is no discernible preference for one method over the others, suggesting a degree of flexibility in method selection within this age group.

This scoping review has some limitations that should be taken into consideration. Firstly, the search process was systematically conducted across three different bibliographic databases (PubMed, Embase, and Web of Science), and the inclusion criteria were limited to studies published in English. While this approach may be perceived as a limitation, as it restricts both language and the number of databases utilized, it is important to note that the search strategy was developed in collaboration with a medical information specialist. This ensured the use of appropriate search terms in each database, thereby enhancing the selection of relevant studies. Additionally, it is worth mentioning that restricting systematic reviews to the English language has been reported to have a limited impact on the effect estimates and conclusions of health-related research [79]. Moreover, utilizing at least two databases has been demonstrated to improve coverage and reduce the risk of missing eligible studies in systematic reviews [80]. Secondly, it is important to note that the full-text evaluation and data extraction processes were carried out by a single reviewer. While this might be perceived as a potential source of bias, it is crucial to highlight that a rigorous and standardized screening protocol was meticulously followed, ensuring transparent inclusion and exclusion criteria for articles. This approach not only promoted consistency but also significantly reduced the likelihood of subjective judgments being introduced. Furthermore, the research team conducted regular discussions and meetings among the authors to align and fine-tune the guideline criteria. This collaborative effort further mitigated the risk of divergent judgments, ultimately enhancing the overall validity and reliability of the study's findings. Finally, due to the nature of this study, we assembled and synthesized a broad and heterogeneous body of evidence. This diversity, stemming from variations in research aims, designs, and methodologies, presents a challenge when attempting to draw direct comparisons between studies and the employed collection methods. A more focused approach might offer the opportunity to provide a more detailed summary of specific methods and better capture their overall effectiveness. Nevertheless, such a limited focus could potentially lead to the omission of crucial data.

It is imperative to emphasize that the primary population under consideration in this study consisted of children and adolescents; however, it is noteworthy that some studies applied variable definitions of the term "adolescent" and included participants who were slightly older. To ensure that the results accurately reflected findings focused on this specific population, the decision was made to include studies that investigated individuals up to 18 years of age. One could argue that such decision would result in the exclusion of relevant literature. However, this decision was made to maintain a more focused and consistent examination of children and adolescents, acknowledging that including older participants might have introduced additional complexities and variables into the study's findings. The inclusion of data solely from healthy individuals is another important point for discussion. While we acknowledge the significance of studying medically compromised children as potential beneficiaries of our research, it is important to recognize that our study did not encompass this group. The reason for that relies on the fact that the current scientific literature lacks a consensus regarding the most appropriate methods for collecting saliva in children and adolescents. In an effort to provide a more focused examination of these methods among healthy individuals, we made the deliberate choice to exclude medically compromised children from our study. This decision aimed to address the pressing need to establish a baseline understanding of saliva collection techniques in a healthy pediatric population. In addition, in cases where participants were taking medication, the type of medication was often not mentioned. Nevertheless, we acknowledge that the exclusion of medically compromised children represents a limitation in the generalizability of our findings, and it underscores the need for future research endeavors to investigate this vital patient subgroup. Despite a substantial number of articles remaining in the review after the selection process (n = 249), the availability of studies directly comparing multiple collection methods within the same population was limited (n = 19). This scarcity of comparative studies restricts our ability to conduct a comprehensive analysis that could provide insights into the relative effectiveness, suitability, and acceptability of these methods. Consequently, the current knowledge base does not fully support a robust understanding of how these collection methods compare. Moreover, it is essential to note that in the majority of the included studies, the primary research focus was not primarily centered on the method of saliva collection itself. In many cases, saliva collection was treated as a peripheral aspect of the overall study, receiving minimal attention and substantiation from the researchers. This prevailing trend underscores a significant gap in comprehensive research that systematically addresses the nuances of different saliva collection methods, especially in the context of various age groups and specific behavioral characteristics. To bridge this gap and enable more informed clinical decision-making, it is strongly recommended that future research efforts employ optimized study designs, such as prospective randomized trials, to delve deeper into the comparative effectiveness and suitability of these methods. Such studies would contribute substantially to advancing our understanding in this critical area.

Nonetheless, the findings of this review provide clinicians with valuable insights to guide their selection of an appropriate saliva collection method tailored to the unique needs of each patient. Overall, the review underscores that saliva collection is generally well tolerated by children and adolescents. However, it is essential to recognize that not every collection method is universally suitable for every age group. Among the collection methods analyzed, the absorption method emerges as the most frequently utilized, followed by the spitting method, the drooling method, and finally, the suction method. Specifically, the suction and absorption methods are favored when dealing with young and/or uncooperative children, owing to their effectiveness and ease of use. In contrast, the drooling and spitting methods are typically only applicable among children aged 3 years and above, given the challenges associated with these methods in younger age groups. To ensure a successful collection procedure, the selection of an appropriate method should be based on the patient's age and developmental level. Notably, in the case of adolescents and older children, there is no discernible preference for one method over another, offering flexibility in method selection within this age group. These insights can greatly assist clinicians in optimizing the saliva collection process for their patients.

5 Conclusion

Overall, saliva collection is well tolerated by children and adolescents, with the absorption and suction methods being preferred in situations involving young and uncooperative children.