Table 2.

Characteristics of included salivary studies (n = 12)

First Author, Year	Sample Type	Storage of Sample	Study Population	Caries Definition	Sample Size	Method of Analysis	Caries-Active Taxa	Caries-Free Taxa
Belstrøm et al., 2017	Stimulated Saliva	Participant was instructed to chew and spit continuously for 3 min into a sterile plastic cup, after which time the collected saliva from the cup was stored at −80°C	Adults ; Ages 18–76; Unspecified	Caries was registered according to Moller and Poulsen as manifest caries on tooth and surface level expressed as DMFT (decayed, missed, filled tooth) and DMFS (decayed, missed, filled surfaces)	n=88; Healthy: n = 57; Caries: n = 31	HOMINGS; bacterial identification was performed using HOMINGS.; V3–V4; Illumina Miseq Sequencing; Statistical Significance: Benjamini-Hochberg correction was used to control for multiple comparisons; For this analysis, an adjusted p-value of <0.0001 was considered statistically significant	Bifidobacterium dentium, Lactobacillus salivarius, Streptococcus sp ot068, Parascardovia denticolens	Fusobacterium periodonticum, Porphyromonas sp ot279, Bergeyella sp ot322, Alloprevotella sp ot914, Stomatobaculum sp ot097, Leptotrichia sp ot223, Lachnoanaerobacul um umeaense
Belstrøm, Paster, et al., 2016	Stimulated Saliva	The subjects expectorated for 1 min, and the saliva was discarded. For an additional 3 min, subjects continued to expectorate, and the saliva was collected in a plastic cup and stored at −80°C.	Adults; Ages 22–70; Unspecified	Dental caries was defined as manifest untreated ≥3 surfaces caries	n=30; Periodiontis: n = 10 ; Dental caries: n = 10; Orally healthy: n = 10	HOMINGS; DNA isolation was performed following specifications of the protocol: Pathogen_Universal_200 (Roche, Mannheim, Germany) (26), and the HOMInGs technique was used for microbial analysis; V3–V4; Illumina Miseq Sequencing; Statistical Significance: p<0.05 in combination with a FDR value of 0 was considered statistically significant	Streptococcus mutans, Streptococcus Genus probe 4, Streptococcus Genus probe 1, Lactobacillus Genus probe 1, L. vaginalis, Actinomyces sp oral taxon 448, Veillonella sp oral taxon 917	NONE FOUND
Dashper et al., 2019	Unstimulated Saliva	Unstimulated saliva, up to 5 mL, was collected from infants using a pipette and from children by passive drooling into a sterile tube. Maternal saliva was collected at the same time as their infant’s second clinical assessment at 7.7 months-of-age. The samples were rapidly frozen at −20 °C, stored and periodically transported to the Melbourne Dental School where they were stored at −80 °C in secure freezers until further analysis.	Children; ages 2 months to 4 years	Caries-Active: ECC as defined as one or more ICDAS II score of 2 or above Caries-Free: no clinically detectable disease	N=134 children were characterized at six time-points from two months up to four years-of-age.	16S rRNA gene sequencing; V4; Ion Amplicon Library Preparation Fusion Method; Statistical Significance: p <0.05	39 months of age Streptococcus mutans 48.6 months-of-age Streptococcus mutans Leptotrichia shahii, Scardovia wiggsiae, Leptotrichia IK040 Increased abundances for development of disease from 39 months-of-age (healthy) to 48.6 months-of age (diseased): Streptococcus mutans, Streptococcus sobrinus, Veillonella parvula	39 months of age Fusobacterium periodonticum, Stomatobaculum longum, Bergeyella 602D02 48.6 months of age Prevotella shahii, Prevotella pallens, Stomatobaculum longum, Porphyromonas CW034 and Capnocytophaga AM20030 Decreased abundances for development of disease from 39 months-of-age (healthy) to 48.6 months-of age (diseased): Prevotella nigrescens, three species of Leptotricia and Actinobaculum 12B759
Eriksson et al., 2018	Supragingival biofilm collected from all accessible tooth surfaces; whole stimulated saliva	Supragingival biofilm was collected from all accessible tooth surfaces with sterile wooden toothpicks and pooled by subject into 100 μL of TE buffer (10mM Tris, 1mM ethylenediaminetetraac etic acid [EDTA], pH 7.6). Whole saliva collected for 3 min into ice-chilled steril test tubes while subjects chewed paraffin wax. All samples were stored at −80 °C.	Adults; Age 17; Unspecified	Numbers of tooth surfaces that had caries in the enamel or into the dentine, had a filling, or were missing were recorded from visual and radiographic examinations. Caries in the enamel was scored according to a visual color change or demineralization within the enamel on bitewing X-rays. Caries in the dentine was scored according to local breakdown in the enamel or a cavity or when demineralization extended into the dentine on bitewing X-rays.	n = 154; 71 present caries (46%); 82 no caries. [This is confusing as the text says 154, but numbers in the table add to 153)	Multiplex 16S rDNA amplicon sequencing with HOMINGS protocol ; V3–V4 ; Illumina Miseq Sequencing; Statistical Significance: P ≤ 0.005	Only S. mutans was significantly (p<=0.005) more abundant in saliva and tooth biofilm samples from subjects with caries than those from caries-free adolescents	NONE FOUND
Gussy et al., 2020	Unstimulated saliva	Unstimulated saliva (1–5 mL) was collected at every wave from infants using a pipette and from children by passive drooling into a sterile tube. Saliva samples were frozen (−20 °C) and periodically transported to laboratory for long term storage at −80 °C.	Children; Ages 2 months-9 years	Caries-Active: d3–6mfs defined as decayed, missing and filled surface index with ICDAS II codes of 3 or greater (i.e. cavitation)	N=419 children were recruited at baseline and were invited to participate in follow-up studies over a period five years of data collection. At baseline, 86 had reported at least one surface tooth cavitation (d3–6mfs)	16S rRNA gene sequencing; V4; Ion Torrent PGM massively-parallel-sequencer; Statistical Significance: p <0.05	Risk for d3–6mfs was significantly higher among children whose saliva sample sequencing over time showed higher percentages of Streptococcus mutans (IRR 1.39, 95 % CI 1.11–1.74, p = 0.005)	NONE REPORTED
Hurley et al., 2019	Unstimulated Saliva and layers of dentinal caries	Caries samples were placed in a sterile 1.5-ml micro-centrifuge tube and transported to the laboratory, where they were frozen until further analysis and stored at −80 °C. Salivary swabs were placed in a collection tube, and stored at−80 °C.	Children; Age <60 months; Primary	For the caries-active group, caries was recorded at the level of cavitation into dentine (cavitation level), using the WHO criteria, with the addition of visible non cavitated dentine caries as referenced by Whelton et al.; Caries-free children did not show clinical evidence of early pre-cavitation of caries or white spot lesions and had no history of treatment on any tooth surfaces	n=138; Severe Early Childhood Caries: n = 68; Caries-Free: n = 70	16S rRNA amplicon sequencing; V4–V5; Illumina MiSeq; Statistical Significance: Benjamini and Hochberg correction; p<0.05	Prevotella histicola, Streptococcus mutans, Veillonella dispar	Alloprevotella tannerae, Haemophilus parainfluenzae, Leptotrichia buccalis, Prevotella salivae
Jiang et al., 2016	Unstimulated saliva	The collected samples were quickly frozen on dry ice, transported to the laboratory within 2 h, and then stored at −80 °C until use	Children; Ages 3–4 ; Unspecified	The caries examination method and criteria recommended by the World Health Organization were followed. Decayed teeth were detected at the cavitation level.	N=40; Caries: n = 20; No Caries: n = 20	16S rRNA gene; V3–V4; Illumina Miseq ; Statistical Significance: p < 0.05	Rothia dentocariosa, Actinomyces graevenitzii, Veillonella sp. oral taxon 780, Prevotella salivae, Streptococcus mutans	Fusobacterium periodonticum, Leptotrichia sp. Oral clone FP036
Luo et al., 2012	Saliva Sample (Subjects expectorated whole saliva)	Subjects expectorated whole saliva into 2-ml sterile Eppendorf microcentrifuge tubes during a 5-min period. All participants were instructed not to clean their teeth the evening and morning before sampling. Two hours before saliva sampling, the subjects were not to eat or drink. Sampling was performed by one examiner. These samples were quick frozen in liquid nitrogen and subsequently stored at −80°C until use	Children; Ages 6–8 ; Mixed	Caries-free: dmfs = 0; Caries-active: dmfs > 8	n = 50; Caries-free: n = 20; Caries-active: n = 30	16S rRNA genes, sampled were assayed using HOMIM microarray; Statistical Significance: p<0.05	Bacteroidetes[G-2] sp _ot274, Capnocytophaga sputigena_ot775, Tannerella sp_ot286, Campylobacter showae_ot763, Campylobacter Cluster I_ot580_748_763, Selenomonas infelix_ot126 and related species ot479_ot481_ot639_ ot054, Parvimonas micra ot111, Leptotrichia hofstadii_ot224	Gemella haemolysans_ot626, Granulicatella elegans_ot596, Streptococcus infantis and sp clone FN042_ot065_638, Streptococcus mitis bv2 and sp clone FP064_ot069_398, Streptococcus sp clone F0042_ot067, Rothia dentocariosa_ot587
Ortiz et al., 2019	Non-stimulated saliva	Non-stimulated saliva specimens (1–2 ml) were collected from the oral cavity of enrolled participants using the OMNIgene-ORAL collection and stabilization kit for microbial DNA (DNA Genotek; OM-501). In some cases, especially for young children, sterile bulbs or sterile swabs were used to collect saliva from the oral cavity, prior to placement of saliva within the OMNIgene-ORAL stabilization reagent. OMNIgene-ORAL stabilizes samples at the point of collection until transport to the laboratory, and permits long-term storage up to 1 year under ambient temperatures.	Children; Ages 2–12; Primary and Mixed	Caries-active: exhibited DMFT scores of 1–15 at the time of appointment. Caries-free individuals: demonstrated no apparent evidence of carious lesions.	n = 85; Caries-Active: n = 64; Caries-Free: n = 21	PCR amplification using V3–V4 16S rDNA-specific primers and next-generation sequencing ; V3–V4 ; Illumina sequencing in the Human Oral Microbial Identification Next Generation Sequencing (NGS) HOMINGS Laboratory ; Statistical Significance: Fold changes larger than 1.5, with the FDR adjusted p-value of < 0.05 were considered statistically significant	Actinomyces gerencseriae, Aggregatibacter actinomycetemcomitans, Alloprevotella tannerae, Bacteroidetes[G-5] Genus probe, Bacteroidetes[G-5] sp HOT 511, Butyrivibrio sp HOT 455, Capnocytophaga haemolytica, Capnocytophaga sp HOT 902, Fusobacterium nucleatum, Lachnospiraceae[G-2] sp HOT 096, Lactococcus lactis, Leptotrichia Genus probe 3, Leptotrichia sp HOT 218, Leptotrichia sp HOT 498, Moraxella Genus probe 2, Mycoplasma salivarium, Neisseria flavescens, Peptococcus sp HOT 167, Peptostreptococcaceae [XI][G-1][Eubacterium] infirmum, Porphyromonas sp HOT 930, Prevotella denticola, Prevotella pleuritidis, Prevotella veroralis, Streptococcus Genus probe 2, Streptococcus sobrinus, Treponema denticola, Treponema Genus probe 4, Treponema lecithinolyticum, Treponema maltophilum, Treponema sp HOT 237, Treponema sp HOT 257	Actinomyces Genus probe 3, Actinomyces johnsonii, Actinomyces lingnae, Actinomyces massiliensis, Actinomyces odontolyticus, Actinomyces sp HOT 170, Actinomyces sp HOT 178, Actinomyces sp HOT 877, Aggregatibacter paraphrophilus, Bacteroidales[G-2] sp HOT 274, Campylobacter concisus, Campylobacter curvus, Campylobacter gracilis, Capnocytophaga Genus probe 2, Capnocytophaga gingivalis, Capnocytophaga leadbetteri, Capnocytophaga sp HOT 332, Capnocytophaga sputigena, Cardiobacterium Genus probe, Cardiobacterium hominis, Cardiobacterium valvarum, Catonella Genus probe, Corynebacterium durum, Corynebacterium Genus probe, Corynebacterium matruchotii, Fusobacterium Genus probe 4, Fusobacterium periodonticum, Gemella Genus probe, Gemella haemolysans, Gemella sanguinis, Granulicatella adiacens, Granulicatella elegans, Haemophilus Genus probe 2, Haemophilus parainfluenzae, Haemophilus pittmaniae, Lachnoanaerobacul um sp HOT 083, Lachnoanaerobacul um umeaense, Lachnospiraceae[G-3] sp HOT 100, Lactobacillus Genus probe 3, Lautropia mirabilis, Leptotrichia Genus probe 4, Leptotrichia sp HOT 219, Leptotrichia sp HOT 223, Leptotrichia sp HOT 392, Oribacterium sinus, Peptostreptococcace ae[XI][G-7] sp HOT 922, Porphyromonas catoniae, Porphyromonas pasteri
Wang et al., 2019	Saliva (To minimize stimulation of salivation, saliva needed to be kept in the mouth for 3 min. Subjects were then instructed to drool into sterile cryogenic vials for 3 min)	Each saliva sample was pipetted into a sterile 1.5-ml Eppendorf tube, which was snap-frozen in liquid nitrogen and stored at −80°C	Children; Ages 3–5 months; Unspecified	Severe ECC: dmfs ≥ 8; Caries-Free: dmfs=0	n = 44; Severe early childhood caries (ECC): n = 25; Caries-free: n = 19	Metagenomics Analysis (gene cataloging); Illumina HiSeq/TruSeq; Statistical Significance: FDR < 0.1	Anaeroglobus geminatus, atopobium rimae, Bifidobacterium dentium, Cryptobacterium curtum, Dialister invisus, Eubacteriaceae bacterium ACC19, Lactobacillus salivarius, Olsenella uli, Oribacterium sp. Oral taxon 078, Parascardovia denticolens, Prevotella amnii, prevotella buccae, Prevotella denticola, prevotella multiformis, Prevotella multisaccharivorax, prevotella nigrescens, prevotella oris, prevotella sp. Oral taxon 317, Shuttleworthia satelles, Streptococcus mutans	Candidatus Nitrospira defluvii, Erysipelotrichaceae bacterium 5_2_54FAA, Neisseria lactamica, Streptococcus australis
Xiao et al., 2018	Nonstimulated whole saliva was collected from subjects through a saliva jet connected to a suction pump at least 2 h after any toothbrushing, eating, or drinking	Previously established methods (Grier et al., 2017; Merkley et al., 2015) were used to perform oral microbiome sequencing and related bioinformatics analysis. Total genomic DNA from clinical samples (100ul saliva and 200ul plaque suspension) was extracted using Quick- DNATM Fecal/Soil Microbe Miniprep Kit (ZymoResearch, Irvine, CA) (Dardas et al., 2014; Merkley et al., 2015; Zhu et al., 2013)	Children; Unspecified: Average Age Children SECC = 4.0 +– 0.9, CF = 3.8 +– 1.6;	S-ECC was diagnosed per criteria of the American Academy of Pediatric Dentistry. The number of carious teeth and surfaces was charted with index variables of decayed, missing due to decay, or filled, according to the codes proposed by the World Health Organization’s Oral Health Surveys Basic Methods (1997). Plaque status for mothers and children were assessed separately according to criteria modified from Silness and Löe (1964) and Ribeiro Ade et al. (2002)	n=39; Severe Early Childhood Caries: 19; Caries-Free: 18	16s rRNA amplicon sequencing; V1–V3 ; Illumina; Statistical Significance: p<0.05	Streptococcus vestibularis, Streptococcus salivarius, Veillonella atypica, Veillonella dispar, Veillonella parvula	Streptococcus ET G 4d04
Yang et al., 2012	Saliva (unspecified)	Two milliliters of saliva were collected from each human-host individual into a tube containing an equal volume of lysis buffer. Samples were stored at −80°C before high-salt DNA extraction	Adults; Ages 18–22	Caries-active: DMFT ≥ 6; Healthy: DMFT=0;	n=45; Healthy: n = 26; Caries Active: n = 19	16s rRNA Amplicon Sequencing; V4–V5; Illumina; Statistical Significance: p < 0.1	Actinomyces sp., Aggregatibacter paraphrophilus, Capnocytophaga granulosa, Catonella morbi, Eggerthella lenta, Enterobacter sakazakii, Fusobacterium periodonticum, Gemella sanguinis, Haemophilus parainfluenzae, Haemophilus sp., Kingella denitrificans, Lachnospiraceae [G-1] sp., Lachnospiraceae [G-2] sp., Lachnospiraceae [G-4] sp., Lachnospiraceae [G-8] sp., Leptotrichia sp., Mobiluncus mulieris, Neisseria sp., Neisseria subflava, Oribacterium sp., Peptococcus sp., Peptoniphilus sp., Peptostreptococcaceae [XI][G-5] sp., Peptostreptococcaceae [XI][G-7] sp., Porphyromonas sp., Prevotella histicola, Prevotella melaninogenica, Prevotella oris, Prevotella sp., Prevotella veroralis, Propionibacterium propionicum, Treponema sp., Treponema vincentii, Veillonella dispar, Veillonella parvula	NONE FOUND