Saliva assay: a call for methodological standardization

Abstract

The oral cavity provides an ideal environment for microorganisms, including bacteria, viruses, and fungi, to flourish. Increasing attention has been focused on the connection between the oral microbiome and both oral and systemic diseases, spurring active research into the collection and analysis of specimens for healthcare purposes. Among the various methods for analyzing the oral microbiome, saliva analysis is especially prominent. Saliva samples, which can be collected non-invasively, provide information on the systemic health and oral microbiome composition of an individual. This review was performed to evaluate the current state of the relevant research through an examination of the literature and to suggest an appropriate assay method for investigating the oral microbiome. We analyzed articles published in English in SCI(E) journals after January 1, 2000, ultimately selecting 53 articles for review. Articles were identified through keyword searches in the PubMed, Embase, Cochrane, Web of Science, and CINAHL databases. Three experienced researchers conducted full-text assessments following title and abstract screening to select appropriate papers. Subsequently, they organized and analyzed the desired data. Our review revealed that most studies utilized unstimulated saliva samples for oral microbiome analysis. Of the 53 studies examined, 29 identified relationships between the oral microbiome and various diseases, such as oral disease, Behçet disease, cancer, and oral lichen planus. However, the studies employed diverse methods of collection and analysis, which compromised the reliability and accuracy of the findings. To address the limitations caused by methodological inconsistencies, a standardized saliva assay should be established.

Graphical Abstract

INTRODUCTION

The oral cavity, which includes the teeth, gingiva, tongue, cheeks, lips, and both hard and soft palates, is open to the external environment. This exposure provides an appropriate setting for microorganisms, offering optimal temperature and humidity for their survival. Consequently, the oral cavity is home to more than 700 to 1,000 species of microorganisms [1]. Moreover, an imbalance in the oral microbiome—known as dysbiosis—is associated with various oral diseases, such as periodontal disease and dental caries. Numerous studies have identified relationships between the oral microbiome and systemic conditions, including cardiovascular disease, pneumonia, heart disease, rheumatoid arthritis, pancreatic cancer, colorectal cancer, stroke, and diabetes [2,3,4,5,6,7,8,9,10]. Understanding the connection between the oral microbiome and systemic diseases is crucial for the diagnosis, prevention, and treatment of these conditions.

Recently, heightened interest in healthcare has prompted active research and development efforts aimed at creating innovative devices and techniques for diagnosis and monitoring [11,12,13]. Concurrently, a growing emphasis has been placed on using patient-friendly and non-invasive samples, such as saliva, sweat, and wound exudate, for these tasks [14,15,16,17,18,19]. Saliva contains a microbiome, various cells, substances derived from the salivary glands, and exogenous materials, as well as blood and its derivatives [20]. This unique composition enables saliva to act as an “ultra-filter” of blood, offering a window into the patient’s overall health and the status of oral microorganisms [21,22].

Lately, the use of saliva for diagnostic purposes has gained prominence, most notably in the detection of severe acute respiratory syndrome coronavirus 2. In a meta-analysis, saliva-based tests demonstrated a sensitivity of 0.88 and a specificity of 0.92 [23]. Additionally, the sensitivity of salivary circulating tumor DNA tests for head and neck cancer has ranged from 47% to 80%. While these results display slight variations depending on the disease, they are considered valuable. A variety of biomarkers, such as mRNA and proteins, can also be utilized in saliva-based diagnostics [24]. Diverse studies are exploring numerous such markers, and ongoing research is expected to further improve diagnostic accuracy [22,25].

However, standard methods for the collection and analysis of saliva samples have not yet been established. Although several studies have used saliva to confirm the presence of oral or systemic diseases, the results vary due to the differing protocols employed for sample collection and analysis, reducing confidence in the findings. A standardized method is needed to ensure the reliability and accuracy of saliva sample collection and analysis. The present review was performed to assess the current state of the relevant research through an examination of the literature and to propose an appropriate assay method for investigating the oral microbiome.

MATERIALS AND METHODS

Search strategy and criteria

This review focused on original research articles relating to saliva and the oral microbiome, published in English in SCI(E) journals between January 2000 and September 2021. The purpose of this investigation was to analyze the scholarly literature on microbiome DNA analysis in saliva samples collected from individuals aged 18 years and older. Consequently, studies outside the scope of this research question were omitted. The exclusion criteria encompassed research on children/adolescents (those ≤18 years old), animals, insects, plants, or specific populations. Additionally, articles lacking data on saliva and those targeting host genomic DNA were not included. Only studies that were verified to have involved 16S rRNA sequencing of microbiome DNA extracted from saliva were included in the review. Studies employing other methodologies, such as polymerase chain reaction and checkerboard DNA-DNA hybridization, were excluded.

The following search terms were used to screen the PubMed, Embase, Cochrane, Web of Science, and CINAHL databases: saliva OR spit OR collect OR acquisition OR submission OR microbiota OR microbial OR microbiome OR microbe OR microorganism OR periphyton OR DNA OR deoxyribonucleic acid. Three experienced reviewers participated in this study, collaborating to design the review and establish the eligibility criteria. Subsequently, the reviewers collectively completed a dual independent review of all records. They began by autonomously screening studies based on their titles and abstracts, then compared their findings. Records were excluded based on reviewer consensus, while those that were not excluded proceeded to full-text screening. When opinions differed, the reviewers jointly reevaluated the record to reach a consensus on whether to exclude it or advance it to full-text screening. Next, the reviewers independently performed full-text screenings of the list of remaining studies, applying the eligibility criteria. Following the completion of these screenings, the results were compared, and records were either excluded or included based on unanimous decision. Disagreements led to a joint reevaluation until a consensus was reached. Additionally, studies lacking the requisite information for data extraction were excluded. Ultimately, studies that met the established criteria were selected for inclusion after full-text assessment.

Data extraction

The following data were extracted from the studies: author(s), year of publication, number of participants, disease focus, environmental factors, intervention applied, age of participants, type of saliva collected, type of collection tube used, sample volume, time of collection, extraction method, and primers utilized.

RESULTS

A total of 353 articles were initially screened based on the specified criteria. Subsequently, 270 articles were excluded after reviewing titles and abstracts, and an additional 30 articles were eliminated following full-text assessment. Ultimately, 53 articles met the inclusion criteria and were selected for the literature review (Figure 1, Table 1) [26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78].

Figure 1. Flow diagram of article selection.

The search terms used were as follows: saliva OR spit OR collect OR acquisition OR submission OR microbiota OR microbial OR microbiome OR microbe OR microorganism OR periphyton OR DNA OR deoxyribonucleic acid. The titles and abstracts of the screened articles were reviewed. Studies that met the established criteria were then selected via full-text assessment.

Table 1. Characteristics of included studies.

Ref.	Type of saliva	Collection medium	Volume	Collection time	Limiting factor	Number of subjects	Disease, environmental factor, intervention	Age
[26]	Whole saliva	Sterile cryogenic vial	2–3 mL	Undistinguished	Refrain from food consumption 8 hr (adult), 45 min (infant)	5 Infant, 5 Primary care givers	Undistinguished	Infant: 4.6 mo, Mother: 30.8 yr
[27]	Passive drooling	Sterilized 50 mL tube	Undistinguished	Undistinguished	Refrain from cleaning teeth with toothpaste or toothbrush, antiseptic mouthwash 14 hrs before 30 min after mouth rinse with water	6 Male, 6 Female	Intervention: probiotic free-diet, commercial probiotic product (Streptococcus thermophiles, Lactobacillus delbrueckii, Lactobacillus paracasei)	Adult
[28]	Unstimulated saliva	15 mL centrifuge tube	5 mL	Undistinguished	At least 2 hr after last meal	15	Disease: AIDS	40–56 yr
[29]	Unstimulated saliva	Sterile 50 mL centrifuge tube	5 mL	Undistinguished	Undistinguished	40	Undistinguished	41.75 yr
	Stimulated additional saliva if needed
[30]	Study: Collection after endoscopy	Undistinguished	2–3 mL	Undistinguished	Before anti-tumor treatment, no prescription of antibiotics 1 mo before	87 ESCC, 63 Dysplasia, 85 Healthy	Disease: esophageal squamous cell carcinoma	ESCC: 64.8 yr, Dysplasia: 65.5 yr, Control: 66.0 yr
	Control: Collection after fasting overnight
[31]	Unstimulated whole saliva	Sterile Eppendorf tube	0.5 mL	Undistinguished	Undistinguished	10 Aggressive periodontitis, 10 Relatives	Disease: aggressive periodontitis	Aggressive periodontitis: 22.1 yr, Relatives: 46.6 yr
[32]	Stimulated saliva	Undistinguished	Undistinguished	5 min paraffin chewing	AM 9 to 11	31 Bechet disease, 15 Control	Disease: Behçet’s disease, intervention, nonsurgical periodontal treatment	Bechet disease: 36.5 yr, Control: 37.8 yr
	5 min paraffin chewing
[33]	Adult: Stimulated saliva	LDTM or OMNIgene	Undistinguished	Undistinguished	Undistinguished	4 Adult, 6 Infant	Undistinguished	Adults: 19–29 yr, Infants: 1–4 yr
	Infant: Swab
[34]	Unstimulated saliva	Sterile Falcon tube	Undistinguished	Undistinguished	Refrain from eating or tooth brushing 1 hr before	61 Fanconi Anemia (53 hematopoietic stem cell transplantation)	Disease: Fanconi Anemia	22 yr (11–44 yr)
[35]	Unstimulated saliva	OMNIgene-ORAL Kit (OM-505; DNA Genotek Inc., Ontario, Canada)	1 mL	Undistinguished	Refrain from eating, drinking, smoking or dental hygiene procedure 30 min before	24	Intervention: short term hospitalization	48.4 yr (19–84 yr)
[36]	Stimulated saliva	Undistinguished	4 mL	Undistinguished	AM 8 to 11	10 Caries, 10 Chronic periodontitis, 10 Control	Disease: chronic periodontitis, caries	Adult
[37]	Stimulated saliva	Undistinguished	Undistinguished	4 min (last 3 min without first 1 min)	AM 8 to PM 2; Before any dental treatment	11 Male, 7 Female	Disease: generalized chronic periodontitis	54 yr (38–75 yr)
	4 min paraffin chewing
[38]	Raw saliva	50 mL Falcon conical tube	2–5 mL	At least 1 min	Refrain from eating, drinking, or rinsing your mouth 1 hr before	41	Undistinguished	25–66 yr
[39]	Unstimulated saliva	50 mL Falcon conical tube	2–5 mL	At least 1 min	Undistinguished	23 Current smokers, 20 Never smokers	Environmental factor: smoking	Current smoker: 34 yr (28–50 yr), Never smoker: 45 yr (33–49 yr)
[40]	Spit	Sterile Falcon tube (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), OMNIgene tube (DNA Genotek Inc.)	1 mL OMNIgene tube (2 mL tube including 1 mL buffer)	Undistinguished	Undistinguished	40	Undistinguished	20–30 yr
	Drooling
	Mouth wash
[41]	Unstimulated saliva	Sterile tube	2 mL	Undistinguished	AM 6 to 8, 30 min after toothbrushing	13 Male, 13 Female	Undistinguished	21–28 yr
	Spit
[42]	Unstimulated saliva	Mouthwash: Scope mouthwash with a 15 wt% alcohol content (Procter & Gamble, Cincinnati, OH, USA)	Whole saliva: 5 mL, Mouthwash: 10 mL	Mouthwash: 30 sec	Refrain from drinking and eating at least 2 hr before	4 Male, 6 Female	Undistinguished	33.5 yr (25–70 yr)
	Mouth wash
[43]	Oral rinse	Sterile graduated test tubes	With 10 mL physiological saline	1 min	AM 9 to 11, refrain from eating and drinking 2 hr before	23 Healthy, 22 Diagnosed with pSS	Disease: primary Sjögren's syndrome	Healthy: 58.0 yr, pSS: 61.5 yr
[44]	Unstimulated whole saliva	Sterile collection tube	Undistinguished	Undistinguished	Before AM 11 in the morning; Refrain from eating or drinking 1 hr before	15 Periodontitis, 20 Healthy	Disease: periodontitis	53.8 yr (Periodontitis: 60.9 yr, Health: 47.3 yr)
	Passive drooling
[45]	Unstimulated saliva	Whole saliva: OMNIgene ORAL OM-505 collection device (DNA Genotek Inc.), Mouth wash: 10 mL of Scope mouthwash	Undistinguished	Mouth wash: 30 sec	Refrain from eating or smoking 20 min	53	Undistinguished	≥18 yr
[46]	Unstimulated saliva	Cryostorage sterile sputum cup	Undistinguished	Undistinguished	Fasting overnight and refrain from tooth brushing; After gargle with 15 mL sterile normal saline for 30 sec	51 Primary bronchogenic carcinoma, 15 Healthy	Disease: bronchogenic carcinoma	Healthy: 56.9 yr, LAC: 54.8 yr, LSCC: 62.4 yr, SCLC: 61.3 yr
[47]	Unstimulated Saliva	OMNIgene saliva collection	Undistinguished	Undistinguished	Fasting	9 High carbohydrate, 10 Low carbohydrate high fat, 10 Periodised carbohydrate	Intervention: dietary intervention	20–35 yr, HCHO: 25.4 yr, PCHO: 27.4 yr, LCHF: 28.3 yr
	Spit
[48]	Unstimulated Saliva	Sterile 1.5 mL microcentrifuge tube	Undistinguished	Undistinguished	Refrain from eating or drinking or brushing 2 hr before; Rinse with sterile water	22 Caries-free, 24 Caries-active	Disease: dental caries	≥60 yr
[49]	Unstimulated saliva	Undistinguished	3 mL	Undistinguished	Refrain from eating and performing oral hygiene procedure at least 2 hr before	Pilot study: Extraction + amoxicllin: 3, Extraction: 3	Intervention: extraction, antibiotics	18–45 yr
						Follow up study: Extraction + amoxicillin: 20, Extraction: 20, Control: 17
[50]	Unstimulated whole saliva	Pre-weighed cup	Undistinguished	Unstimulated saliva : 15 min	Refrained from eating, drinking, and smoking 1 hr before	15 Sjögren's syndrome, 15 Dry mouth, 15 Healthy	Disease: Sjögren's syndrome/dry mouth	30–80 yr, Sjögren's syndrome: 53.2 yr, Dry mouth: 53.1 yr
	Stimulated whole saliva with chewing 5 min (paraffin wax table, Ivoclar Vivadent, Schaan,Liechtenstein)							Healthy: 56 yr
[51]	Whole unstimulated saliva	Undistinguished	2 mL	Undistinguished	Refrain from consuming food and drink more than 2 hrs before	39 Alzheimer’s disease, 39 Control	Disease: Alzheimer’s disease	Alzheimer's disease: 65 yr, Control: 63.4 yr
[52]	Unstimulated saliva	50 mL sterile tube	1 mL	Undistinguished	Refrain from eating or drinking 1 hr before	15 Healthy (5 male, 10 female), 15 CG patients (7 male, 8 female)	Disease: Cheilitis granulomatosa	Healthy: 53.7 yr, CG: 47.6 yr
[53]	Unstimulated Saliva	COPAN eNAT (COPAN group, Brescia, Italy)	Undistinguished	20 sec	Mother: Restrain from flossing, tooth brushing, or using mouth rinse 12 hr before, Restrain from drinking fluid without water, eating, smoking, or using chewing gum 2 hr before	30 Mothers, 30 Infants	Undistinguished	Mothers: 32.3 yr (25–41 yr), Infants: 7.5 mo (2–15 mo)
					Infant: Restrain from feeding, tooth brushing, and breast feeding 30–60 min before
[54]	Fresh saliva	Sterile graduated test-tube	Undistinguished	Undistinguished	Refrain from tooth brushing from the night, eating and drinking 2 hrs before; Rinse mouths thoroughly 3 times 3 min with 10 mL scope mouthwash	55 Reflux esophagitis, 51 Healthy	Disease: reflux esophagitis	Reflux esophagitis: 52.6 yr, Control: 48.9 yr
[55]	Passive drooling, Unstimulated forepart saliva	Graduated polypropylene tube (Eppendorf, Hamburg, Germany) through funnel (Zhenqi, Shanghai, Chaina)	1.5 mL	Undistinguished	AM 9 to 10; Refrain from eating, tooth brushing, drinking, exercising, or chewing gum 2 hr; Rinse mouth with deionized water, Rest 10 min (sitting upright)	5 Female, 5 Male	Intervention: different stimulating conditions	≥18 yr
	Passive drooling, Unstimulated midstream saliva
	Olfaction stimulated forepart whole saliva with fresh lemon juice
	Olfaction stimulated midstream saliva
	Stimulated forepart whole saliva with citric acid swab
	Stimulated midstream saliva with citric acid swab
	Stimulated forepart saliva with paraffin gum
	Stimulated midstream saliva with paraffin gum
[56]	Unstimulated whole saliva	Undistinguished	2 mL	Undistinguished	AM 8 to PM 12; Refrain from eating and drinking	10 Erosive LP, 10 Non erosive LP, 10 Healthy, 10 Recurrent aphthous ulceration	Disease: LP	Erosive LP: 51.6 yr, Non erosive LP: 46.6 yr, Healthy: 47 yr, Ulcer: 34.7 yr
[57]	Unstimulated saliva	50 mL collection tube (Becton, Dickinson and Company)	10 mL	Undistinguished	Refrain from tooth brushing the night before and the morning of sampling, and having breakfast; At AM 8, gargle mouth with sterile saline	10 PC, 17 BPD, 10 Healthy	Disease: pancreatic cancer, benign pancreatic disease	PC: 57.4 yr, BPD: 42.8 yr, Healthy: 31.1 yr
	Spit
[58]	Stimulated saliva	50 mL conical BD Falcon polypropylene centrifuge tube (Corning Inc., Corning, NY, USA)	Undistinguished	2 min	Undistinguished	27 Healthy, 39 Blood cancer (16 AML)	Disease: hematological cancer diagnoses, 3 ALL, 16 AML, 1 CML, 10 LYM, 2 MDS, 2 MF, 5 MM	Healthy: 53.2 yr, Blood cancer: 52.2 yr, AML: 53.2 yr
	Chewing unsweetened and unflavored gum (The Wrigley Company, Mars, Inc., Chicago, IL, USA) for 2 min
[59]	Unstimulated saliva	30 mL sterile conical tube	Undistinguished	1 min	Refrain from eating or drinking 30 min	10	Disease: cystic fibrosis, stage Early/Int/Adv	32 yr (19–56 yr)
	Drooling
[60]	Unstimulated saliva	DNAse free plastic tube	Undistinguished	1 min	Undistinguished	12	Disease: acute endodontic infection	No anti: 37.5 yr (22–56 yr), Anti: 44.3 yr (40–53 yr)
							intervention, antibiotics
[61]	Saliva	Oragene DNA OG-500 collection kits (DNA Genotek Inc.)	Undistinguished	Undistinguished	Refraining from eating or drinking after midnight	10 Chlorhexidine, 10 No treatment	Intervention: 0.12% chlorhexidine mouth rinse twice daily for 2 week before endoscopy	≥18 yr
[62]	Unstimulated Saliva	50mL sterile tube	2 mL	Undistinguished	Refrain from drinking and eating 2 hr and cleaning their mouth of obvious food residue and teeth	15 OSAS, 9 Control	Disease: OSAS	OSAS: 47 yr, Control: 40.2 yr
[63]	Unstimulated saliva	50 mL Sterile tube	Up to 2 mL	Undistinguished	AM 9 to PM 4	1,349 at 7 Community Support Centers in Miyagi Prefecture	Undistinguished	54.9 yr (45–90 yr)
[64]	Unstimulated saliva	Undistinguished	3 mL	Undistinguished	Refrain from ingestion, rinsing, and oral hygiene measure 1 hr before	4 Periodontally healthy, 8 Chronic periodontitis	Disease: chronic periodontitis	Periodontally healthy: 41 yr (26–52 yr), Chronic periodontitis: 52 yr (38–58 yr)
[65]	Unstimulated saliva	50 mL collection tube (Becton, Dickinson and Company)	2–5 mL	Undistinguished	Refrain from dieting, smoking, and oral hygiene prophylaxis 2 hr before	10 Gingival SCC, 15 Periodontitis, 15 Healthy	Disease: gingival SCC, periodontitis	38–73 yr, Gingival SCC: 61 yr, Periodontitis: 60 yr, Healthy: 60.9 yr
[66]	Stimulated saliva	Undistinguished	Undistinguished	Undistinguished	Refrain from eating, drinking, tooth brushing or smoking 1 hr before	16 Study, 16 Control	Intervention: stannous and fluoride ions containing dentifrice and mouth rinse	Study: 35.8 yr, Control: 34.9 yr
	Sterile paraffin chewing
[67]	Passive drooling	10 mL Techno Plas sterile centrifuge tube	3 mL	Undistinguished	At awake; Refrain from eating or drinking	40 Control, 40 Depression/anxiety, no IBS, 40 IBS, no depression/anxiety, 40 Depression/anxiety, IBS	Disease: depression, anxiety, IBS	18–40 yr
[68]	Stimulated saliva	Salivette	1 mL	4 min (last 3 min without first 1 min)	AM 6 to 9; Refrain from food or drink consumption and their oral hygiene routine	20 Sucrose & probiotic, 20 Sucrose & placebo, 20 Xylitol & probiotic, 20 Xylitol & placebo	Intervention: sugar, probiotic lozenge (Lacticaseibacillus rhamnosus & Latilactobacillus curvatus)	20–32 yr
	4 min paraffin chewing
[69]	Unstimulated saliva	15 mL collection tube (catalog number 352057; Corning Inc.)	Undistinguished	5 min	Undistinguished	38	Undistinguished	30.8 yr
[70]	Saliva	OMNI gene-oral 501 tube	Undistinguished	Undistinguished	In the morning; Before brushing tooth, eating or drinking	10 Lean, 9 Obese	Intervention: fermented soy beverage (Q-CAN contained 8% fermented soy powder)	32 yr
	Spit
[71]	Saliva	50mL sterile tube	Approximately 10 mL	Undistinguished	Refrain from food, smoking, or chewing gum 2 hr before	84 High altitude (<3,650 m) Tibetian, 83 Ultra-high altitude (>4,000 m) Tibetian	Environmental factor: altitude	High altitude Tibetian: 19 yr, Ultra-high altitude Tibetian: 19 yr
[72]	Saliva	Sterile sample container	Undistinguished	5 min	Refrain from eating, drinking, tooth brushing, or gum chewing	12 E-cigarettes, 12 Control	Environmental factor: E-cigarette	18–30 yr (Control: 21 yr, E-cigarette: 21 yr)
	Drooling				After rinse with water
[73]	Unstimulated saliva	Undistinguished	2–3 mL	Undistinguished	Refrain from eating or brushing tooth 1 hr before	81	Disease: dental and periodontal health status	25–65 yr
	Spit
[74]	Saliva	OMNIgene OM-501 (DNA Genotek Inc.)	Undistinguished	Undistinguished	Undistinguished	52	Disease: pancreatic diseases or gastrointestinal disease	64.1 yr
[75]	Resting whole saliva	OMNIgene DIsvocer Kit 505 (DNA Genotek Inc.)	At least 5 mL	Undistinguished	Refrain from eating, smoking and dental procedure 1 hr before	25 Healthy, 25 Crohn’s disease	Disease: Crohn’s disease - Controlled with Crohn’s disease Activity Index scores lower than 150	Healthy: 40 yr (21–66 yr), Crohn’s disease: 40 (23–67 yr)
[76]	Unstimulated saliva	50 mL Falcon conical tube	2–5 mL	At least 1 min	AM 8 to 10; Refrain from drinking, smoking, or oral hygiene procedure 1 hr before	20 Abnormal, 20 Normal	Disease: serum thyroid-stimulating hormone levels (4.2 mIU/L)	45–60 yr
[77]	Pure saliva	Sterile centrifuge tubes	2 mL	Undistinguished	Refrain from eating 2 hr before	20	Undistinguished	20–30 yr
[78]	Unstimulated whole saliva	OMNIgene ORAL (OM-501; DNA Genotek Inc.)	Undistinguished	Undistinguished	Refrain from eating, drinking, smoking, gargling, or tooth brushing 2 hr before	21 Palmoplantar pustulosis, 10 Healthy	Disease: Palmoplantar pustulosis	Palmoplantar pustulosis: 46.6 yr, Healthy: 35.5 yr

AIDS: acquired immunodeficiency syndrome, ESCC: esophageal squamous cell carcinoma, LAC: lung adenocarcinoma, LSCC: lung squamous cell carcinoma, SCLC: small cell lung cancer, LP: lichen planus, PC: Pancreatic cancer, BPD: Benign pancreatic disease, AML: acute myelogenous leukemia, ALL: acute lymphoblastic leukemia, CML: chronic myelogenous leukemia, LYM: lymphoma, MDS: myelodysplastic syndrome, MF: myelofibrosis, MM: multiple myeloma, OSAS: obstructive sleep apnea syndrome, SCC: squamous cell carcinoma, IBS: irritable bowel syndrome.

Saliva sample collection and analysis methods varied across the studies, with some failing to report the sampling and analysis techniques used. Notably, 10 articles referred to the samples as either whole saliva or pure saliva without specifying the sampling method. The most commonly used method was unstimulated saliva collection (31 of the 53 studies), followed by stimulated saliva collection (10 of 53), and mouthwash (3 of 53) (Table 2). Passive drooling (7 of 53 studies) and spitting (6 of 53) were also employed to obtain unstimulated saliva (Table 2). Two studies utilized mouth swabs for sample collection, but as these were specific to astronauts within a specific community [79,80], they were excluded from the review.

Table 2. Classification according to type of saliva, collection methods, and characteristics of subjects.

Classification		No.
Type of saliva
	Unstimulated saliva	31
	Stimulated saliva	10
	Mouth wash	3
Collection method
	Passive drooling	7
	Spit	6
	Mouth swab	0
Characteristics of subject
	Disease	29
	Intervention	11
	Environmental factor	3

Among the studies, 29 identified relationships between various diseases and the oral microbiome, with most focusing on dental conditions such as periodontal disease and dental caries. Other articles examined cancer and oral soft tissue diseases, including Behçet disease and oral lichen planus. Furthermore, 11 articles investigated the impact of interventions, including probiotics and anabolic agents, on the oral microbiome. Three articles examined the influence of environmental factors, such as smoking, while 9 articles evaluated or compared different methodologies for collecting and storing oral samples, including saliva (Table 2).

Most studies utilized either Falcon conical tubes or Eppendorf tubes to collect saliva samples, although some described the use of collection kits or media. Sample volume and collection time varied across studies, with some articles not reporting these data (Table 1). Although DNA extraction was predominantly performed with Qiagen kits, the specific methods differed among the studies. The primers used for DNA amplification mainly targeted the V3-V4 region of the 16S rRNA, but variations were noted in this practice as well (Supplementary Table 1). Participants were instructed to abstain from eating, drinking, and performing oral hygiene before providing saliva samples; however, the protocols regarding fasting time, collection restrictions, and other parameters also varied from study to study (Table 1).

DISCUSSION

In this review, we detail various methodologies for collecting and analyzing saliva samples to study the microbiome. We also aimed to compare changes in the oral microbiota based on disease and intervention. However, this comparison was precluded by substantial variation in collection and analysis methods depending on the participant or study approach. A standardized method is essential to ensure the reliability and comparability of future studies.

Drug intake tends to increase with age, leading to a reduction in saliva secretion. Additionally, conditions affecting the salivary glands, such as Sjogren syndrome or sialolithiasis, result in diminished saliva production. Treatments like radiation therapy or chemotherapy for head and neck cancers are also known to decrease saliva secretion [81,82]. In this review, most of the studies utilized unstimulated saliva samples for their assays. However, collecting an adequate volume of unstimulated saliva from patients with compromised secretion can be challenging. Therefore, an appropriate saliva collection method must be established to promote patient convenience and to improve the reliability of the results.

Saliva can be classified as either stimulated or unstimulated, depending on whether external stimulation is applied. As previously noted, unstimulated saliva is commonly used. However, collecting adequate saliva samples can be challenging depending on the patient’s health status and level of cooperation. Consequently, 10 of the studies in this review utilized stimulated rather than unstimulated saliva. Importantly, unstimulated and stimulated saliva differ in their composition of both inorganic and organic components. Additional research is essential to determine which type of saliva more accurately reflects an individual’s overall health status, encompassing both systemic and oral health [83,84,85,86]. Furthermore, the mouthwash technique was used in 3 studies in this review. According to recent findings from our research group, in the context of oral microbiome analysis, the results obtained with mouthwash are not inferior to those collected through passive drooling of unstimulated saliva in terms of convenience and effectiveness [87]. Similarly, while a study conducted by Maruyama et al. did not focus on the microbiome, their results produced with the mouthwash method were comparable to those obtained with unstimulated saliva. Therefore, the mouthwash technique may be a viable alternative for saliva research, although further validation is necessary [88].

The collection methods for unstimulated saliva can be broadly categorized into passive drooling and spitting. Passive drooling, in use since 1934, was previously considered the gold standard for obtaining samples with minimal impact on saliva composition. However, this approach relies on patient cooperation, and it is challenging to implement in individuals with decreased saliva secretion [21,89]. Compared to drooling, spitting offers the advantage of rapid collection, even in those with relatively low saliva secretion. However, spitting may induce stimulation and subsequently influence salivation, leading to variations in results that must be considered [21,90]. Additionally, a mouth swab is available for use in patients with limited cooperation. When appropriately applied, this method enables the collection of saliva without inducing stimulation [21]. None of the papers included in this study employed the mouth swab technique; this is partially because the research targeted adults, obviating the need for cooperation from children. Nevertheless, additional research is imperative to explore the viability of the mouth swab method for adults who may exhibit limited cooperation, such as patients in the intensive care unit.

Most of the examined studies required patients to abstain from eating, drinking, and brushing their teeth for approximately 1 to 2 hours prior to saliva collection. Additionally, sample collection typically occurred between 9:00 and 11:00 AM, although the exact timing varied (Table 1) [26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78]. A review article by Aripin et al. [91] suggests that fasting can influence various factors, including salivary levels of inorganic compounds and total protein, most notably altering salivary nitrite concentrations. Their study focused on the systemic effects of changes in nitric oxide levels and their potential implications for the diagnosis and treatment of coronavirus disease 2019 (COVID-19) [91]. However, factors other than fasting may also impact the composition and quantity of oral microbes. Consequently, further research in this area is warranted. The oral cavity is not an isolated environment; it is open to the external environment and is therefore susceptible to influences from activities, like eating and drinking. This makes it challenging to compare results across the included studies, lowering the confidence in such comparisons. To address this issue, a standardized method for saliva collection should be established.

For precise assessment of changes in the oral microbiota, as well as diagnosis and disease monitoring, the entire oral microbiota must be collected and preserved. However, factors such as collection methods, storage conditions (such as temperature), and the choice of preservation media can potentially influence the results. Several studies have explored the effects of different storage conditions, preservation kits, and media on the oral microbiome [87,92].

In the present review, most studies employed Qiagen DNA extraction kits; however, the specific kits varied across studies. Notably, composition, components, and methodologies may differ even among Qiagen products. Furthermore, some studies utilized extraction kits or methods from manufacturers other than Qiagen. The potential influence of these variations on the results of oral microbiome analysis must be considered.

Taxonomic identification using 16S rRNA is a common approach for analyzing the oral microbiome. However, 16S rRNA includes several variable regions, labeled V1-V9. The V3-V4 region is frequently targeted for next-generation sequencing, and most studies have sequenced this region. The choice of 16S rRNA variable region depends on the research objectives; nonetheless, selecting a particular region is essential for establishing a standardized methodology.

Most of the articles in this review were cross-sectional in nature. However, the oral cavity and its microbiome are influenced by various factors; therefore, a longitudinal study design may be required to produce findings with high reliability and accuracy [2]. This necessitates the development of a longitudinal research framework, along with a standardized collection method that supports the long-term storage of samples. Furthermore, as a disease progresses, changes in both the host and the microbiome may occur. To ascertain the precise causality rather than merely the association between the disease and the microbiome, it is important to analyze genomic, transcriptomic, proteomic, and metabolomic data [2]. Consequently, a collection method must be established that enables the acquisition of the necessary data while minimizing patient inconvenience.

Furthermore, appropriate and tailored assay methods must be developed to detect fungi, viruses, and archaea in the oral cavity. Recent research has leveraged saliva as a rapid and minimally invasive diagnostic tool for identifying various bioanalytes, thereby revealing the presence of various organisms within the microbiome. Notably, saliva has been utilized to detect COVID-19 [93,94]. Although bacteria constitute the majority of the oral microbiome, other microorganisms, including fungi and viruses—which comprise only about 0.1%—can also impact host health. These organisms influence the composition and growth of the oral microbiome; thus, their roles should be clarified in future studies [95,96,97]. Despite their relatively small proportion, the composition and abundance of these microorganisms vary depending on an individual’s systemic health and infection status. To date, studies in this area have been limited and often overlooked. Nonetheless, given the potential effects of microbial fluctuations on the oral microbiome, oral diseases, and systemic health conditions, continued research is crucial [98,99,100]. Consequently, the standardization of saliva assays targeting the microbiome is imperative to improve diagnostic accuracy and reliability.

Studies targeting infants or children, which did not meet the inclusion criteria for this study, similarly employed varied methods for collection and analysis. Given that infants are less cooperative than adults, saliva collection was performed using techniques such as swabbing or sponging. Accordingly, it is also essential to standardize saliva assays in infants to promote reliability, accuracy, and comparability across studies.

This review has certain limitations. Most notably, we were unable to analyze and compare results by disease or intervention—an original objective of the review—due to variations in the methodologies employed.

CONCLUSION

In conclusion, many studies have employed saliva to characterize the oral microbiome in the context of specific diseases, interventions, and environmental factors. However, the methodologies employed for saliva assays are diverse, undermining the reliability of the findings and hindering comparisons across studies. To overcome these limitations, a standardized saliva assay protocol must be established.

SUPPLEMENTARY MATERIAL

Supplementary Table 1

Extraction method(kit), Primer/PCR and analysis method of included studies.