Introduction

Periodontal diseases refer to inflammation of the gingival tissue surrounding the teeth and the supportive structures of teeth that are highly prevalent.1,2,3 Inflammation of the gingival tissue (gingivitis) arises when dental plaque accumulates along the gingival margin due to poor oral hygiene.4,5 If not treated, gingivitis can progress to periodontitis, which is distinguished by destruction of supporting connective tissue, alveolar bone loss, and ultimately result in tooth loss.6 Periodontitis is a multifactorial disease with complex pathogenesis. Although microorganisms are the main etiological agents, genetic predisposition and environmental factors, such as smoking, can alter the host immune-inflammatory response.7,8,9,10,11,12,13,14,15,16,17,18 Gingivitis is common in the United States with reports indicating upwards of 50% of the adult population had gingivitis.19,20 A survey of adults in the United Kingdom estimated that 42% of 35–44 years old and 70% of 55–64 years showed evidence of periodontitis,21 and similar results were found in American adults.22

Treatment of plaque-induced periodontal disease begins with controlling the biofilm by professional oral hygiene cleaning, scaling, and root planning, along with oral hygiene instructions.23,24,25,26 In cases where controlling the plaque is not sufficient or in aggressive disease form, the treatment options include the use of systemic antibiotics or periodontal surgery to gain further access to the root for debridement, alongside procedures to regenerate lost tissues.27,28,29,30,31 Successful treatment for periodontitis may reduce inflammation and regenerate some of the supporting bone and connective tissues. While gingivitis is preventable and reversible with proper oral hygiene, periodontitis is an irreversible condition since a complete restoration of the lost tooth and support is impossible.9

Common symptoms for periodontitis are spontaneous bleeding, loosening of teeth, sporadic pain and discomfort. However, both patients and dental practitioners underestimate the disease since it can be painless and asymptomatic, and thus go unnoticed and untreated for years. Noting that the bone loss associated with periodontitis is irreversible makes it imperative for oral health providers to identify periodontal disease progression as soon as possible to minimise adverse health effects. Currently, progression of periodontal disease from gingivitis to periodontitis is not well described, as clinicians often use criteria that rely on measurements that are error-prone and classifications that require the knowledge of the rate of disease progression where this may be unavailable.32,33,34,35 Despite the advancements made in the pathogenesis of periodontal disease, most diagnoses are still based almost entirely in traditional clinical assessment criteria that include presence or absence of bleeding upon probing (BOP), probe pocket depth,32 clinical attachment loss (CAL), and the patient's medical and dental histories.36,37 However, this visual method of evaluating periodontal disease may only state the presence or absence of periodontal disease. It leaves no opportunity for the ability to predict future diseases, to determine the underlying cause of a present disease, or to determine the appropriate treatment plan for each individual. With the growing use of genomics, proteomics, and bioinformatics in medicine, noninvasive methods for disease diagnosis are attractive endeavours.

The field of salivary diagnostics to allow risk determination for both oral and systemic diseases is advancing. Researchers are looking into the use of saliva as a diagnostic medium that would be able to aid clinicians in risk determination, diagnosis, and treatment planning for periodontal diseases.38,39,40,41,42,43

Saliva as a diagnostic medium

Saliva is a promising target for diagnostic tests as its collection is noninvasive and it is readily available. Biomarkers for the detection of diseases, such as caries, oral cancer and periodontal disease, as well as systemic disease such as hepatitis, Sjögren's syndrome, breast cancer, pancreatic cancer and HIV have been shown to be present in saliva.44,45,46,47,48,49,50,51 Whole saliva can be obtained by either unstimulated or stimulated collection. Stimulated saliva collection is accomplished by masticatory or gustatory stimulation, for example, chewing on parafifin or placing citric acid on the patient's tongue. Unstimulated saliva collection occurs when there is no masticatory, gustatory, or mechanical stimulation and is mainly affected by patient hydration level.52 Whole saliva contains a mixture of fluids from the major and minor salivary glands, gingival crevicular fluid (GCF), serum, immune and epithelial cells, and many microbes.53 A large body of scientific research has focused on GCF biomarkers that communicate site-specific periodontal disease progression; however, it is more difficult to implement clinically due to the possibility for salivary contamination, difficulty to probe all tooth sites and potentially statistical method error.54,55,56,57,58 Therefore, this review will focus on advances in salivary biomarkers for periodontal disease, as all components of whole saliva are analysed without risking local fluid contamination.

Current detected biomarkers for periodontal disease

A wide variety of classes of biomarkers are found in saliva including proteins of host and bacterial origin; DNA and mRNA of host, bacterial, and viral origin; ions and steroid hormones.59,60,61 Papers since 2010 have shown that microbial, genetic damage and protein biomarkers obtained from saliva are informative in the detection of gingival inflammation and periodontal disease activity (see Table 1). These recent studies add to an already existing wealth of research that has shown and confirmed detectable biomarkers for periodontal disease in saliva. For many biomarker studies the area under the receiver operating characteristics (ROC) curve (AUC) is an important measurement to report the performance of a biomarker by indicating whether the biomarker can discriminate between individuals with and those without the disease. The measurement ranges from 50%, representing a test no better than chance, and 100%, representing a perfect diagnostic test.62,63

Table 1 The results of tested biomarkers for periodontal disease and the clinical parameters used in some recent publications. In addition to p-value, area under curve (AUC) values are presented when provided
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Matrix metalloproteinases (MMPs) are zinc-dependent proteases that are known to be associated in diseases such as arthritis, atherosclerosis, as well as periodontitis, because they are involved in the degradation of various extracellular, pericellular and non-matrix substrates.64,65,66 MMPs are regulated by a family of endogenous inhibitors, called the tissue inhibitors of metalloproteinases (TIMP) and the ratio of MMP/TIMP has been a useful measurement to identify potential imbalances between synthesis and degradation as an indicator of periodontal disease.67,68 In particular, MMP-8 has been known to be a biomarker for inflammatory and periodontal diseases.69,70,71 Recent studies further confirmed this association. In a Finnish population, MMP-8 in saliva was tested by two detection methods, immunofluorometric assay (IFMA) and enzyme-linked immunoassay (ELISA), and it was found that detection of periodontitis subjects from controls was stronger by IFMA than ELISA (area under the ROC curve (AUC) reported as 0.751 and 0.592, respectively).72 The study also found that utilising MMP-8 with one other marker, either pyridinoline crosslinked carboxyterminal telopeptide of type I collagen (ICTP) or TIMP-1, increased the AUC for non-smoking groups. The AUC values were reported as 0.819 and 0.817 for the protein combinations in non-smoking subjects. MMP-8 was further researched in a Swedish population and this study confirmed it was able to significantly discriminate severe periodontitis over less advanced periodontitis (p <0.001), showing that MMP-8 is also able to function as a marker for periodontal disease activity.73 In the same study, interleukin-1β (IL-1β) and the protein combination ratio of MMP-8/TIMP-1 was higher in severe periodontitis patients (p <0.001).

A study conducted at the University of Kentucky reported that MMP-8 was higher in chronic adult periodontitis patients than healthy controls (AUC: 0.92).74 The study also showed this same high detection capabilities in IL-1β and interleukin-6 (IL-6) (AUC: 0.95 for both) and when these three protein markers were combined the AUC elevated to 0.984 with a sensitivity of 0.94 and specificity of 0.966. MMP-9, previously shown to increase with periodontal disease severity,75 and TIMP-1 was studied in a Colombian population. Significantly higher levels of both MMP-9 and TIMP-1 were seen in chronic periodontitis subjects compared with healthy controls (p values were <0.001 and 0.010, respectively) and when used in combination, significance was also shown between the two groups (p <0.001).76

Other protein biomarkers in saliva for periodontal disease have recently been studied. Quantitative proteomics was analysed to determine the alterations in the salivary proteome before and after periodontal treatment was administered.77 The prominent findings were for proteins S100A6, S100A8, and S100A9 where abundance increased by fold changes of 1.64, 2.31, and 1.99, respectively. Levels of S100A8/A9 have previously been shown in GCF and saliva to correlate with periodontitis likely due to active secretion by gingival keratinocytes and neutrophils that are infiltrating.78,79 However, S100A6 has little reports associating the protein to inflammation. One study showed an upregulation of S100A6 in a mouse model of asthma.80 Noting that the S100 proteins increased in abundance during the disease inactive state highlights the involvement in the host response during periodontitis. These may be potential biomarkers for monitoring periodontal disease activity. Macrophage inflammatory protein-1α (MIP-1α), an upstream signalling molecule associated with bone resorption by osteoclasts, was recently studied at the University of Kentucky.81 Results found that MIP-1α can significantly detect periodontal disease from controls (AUC: 0.94). Lactoferrin, a metalloprotein found in exocrine secretions that has previously shown correlations with chronic periodontitis in GCF,82 was also investigated in a Swedish study.83 The study showed an association between chronic periodontitis patients and increased lactoferrin concentration compared with healthy controls in saliva (p <0.05).

Markers of DNA damage that are excreted in bodily fluids can also be useful in diagnostics as they are evident of active DNA repair.84

8-Hydroxydeoxyguanosine (8-OHdG) is an oxidized nucleoside that is commonly used as a marker for oxidative DNA damage in inflammatory diseases.85,86,87 8-OHdG was further studied in saliva to determine its potential as a marker for periodontitis disease activity in a Turkish population. Results found that 8-OHdG levels of the chronic periodontitis group were statistically higher than healthy and chronic gingivitis subjects (p <0.001). This may be useful as a marker for periodontal disease activity as it is also correlated with PD and CAL (p <0.001) that are used as disease severity parameters.88

Bacteria in saliva have also been recently confirmed to be biomarkers for periodontitis. Previous studies have shown the relationship between salivary microorganisms and periodontal disease.7,89 A recent study aimed to determine if microbe salivary copy-count could be utilised to distinguish between individuals of different periodontal health classifications. The results showed that three bacterial species, Porphyromonas gingivalis, Tannerella forsythia and Prevotella intermedia, could identify periodontitis groups from healthy and gingivitis subjects with high sensitivity and specificity solely on their salivary copy-counts by Taq-man real-time PCR. This data suggests that using saliva microbial copy-counts may be useful in a clinical setting to determine individuals with periodontitis; however, more studies need to be conducted to be able to distinguish the different subtypes of periodontitis.90

Currently, there is no single biomarker that is specific to identify periodontal disease. Therefore, the discovered salivary biomarkers from microbial and host origins can be used in combination to increase the specificity for diagnosis of current periodontal and future disease progression in a clinical setting.60,91 There are two commercially available salivary diagnostic tests for the detection of periodontal diseases.92 The first, MyPerioPath®, identifies the species and concentration of salivary bacteria that are associated with gingivitis and periodontitis, thus supporting the clinician to identify future risks and developing personalised treatment options for the best targeted care.93,94 It has been shown that the presence of multiple pathogenic periodontal bacterial species is more closely associated with periodontitis than the presence of any one species.89 The second, MyPerioID®, determines a patient's genetic susceptibility to periodontal disease because of their increased production of the inflammatory cytokines interleukin-1 α and β (IL-1 α and IL-1 β) during an inflammatory response.42,95,96,97 This test is scientifically based on genetic polymorphisms of these two genes that increase the production of interleukin-1, a known regulator of the inflammatory response.12,98 Both tests were developed by OralDNA® Labs and are currently available through dental care practitioners who are given detailed result reports that can be used to supplement traditional assessments and shared with the patient to inform them of their oral health. However, a shortcoming of these diagnostic tests is that four to five days are needed for results to be delivered. Also, they are able to identify risk factors for periodontitis, but they lack the ability to determine disease activity and provide a projected timeline for when periodontal attachment loss and bone resorption will occur.95

Point-of-care devices for salivary diagnostics

The goal of salivary diagnostics is to be able to provide information regarding a number of oral and systemic disease status results to clinicians and patients during the time of a regular check-up. A main hindrance has been that many biomarkers are available in very low quantities in saliva, therefore making detection sensitivity a challenge. However, there are currently different groups working on point-of-care (POC) devices that will allow quick and accurate results using increasingly sensitive detection mechanisms.99 A group at the University of Texas at Austin has applied an electronic microchip-assay to detect C-reactive protein (CRP), a biomarker for inflammation associated with periodontal disease at the picogram per milliliter level.100 CRP is a systemic marker produced as a response to inflammatory stimuli101,102 that can differentiate between healthy and periodontitis in serum103,104,105,106 and saliva.44,107 As CRP is available in lower concentrations in saliva compared to serum, the increased sensitivity of the microchip made it a reality to distinguish between healthy periodontium and chronic gingival inflammation based directly on salivary CRP levels.44,100 However, CRP is a systemic marker of inflammation that has been shown to significantly increase for other conditions, including myocardial infarction, atherosclerosis and arthritis.108,109,110 Researchers at Sandia National Laboratories and the University of Michigan, Ann Arbor have also established a POC device that is able to perform immunological assays in under ten minutes with low sample volume and concentration requirements to test for periodontal disease. This device iscalled the integrated microfluidic platform for oral diagnostics (IMPOD).99 The IMPOD is able to detect proteins in the picoMolar range for necrosis factor-α (TNF-α) and interleukin-6 (IL6) that has been spiked-in to saliva. TNF-α, a proinflammatory and immune regulatory cytokine, has been identified in saliva and is significantly elevated in people with periodontitis compared with healthy individuals, with increased levels being correlated with increased number of sites with bleeding upon probing, pocket depth, and higher clinical attachment levels.111,112,113,114 IL-6, which is released in response to IL-1 and TNF, has been shown to increase proportionally with bone loss in adult chronic periodontitis patients.115,116 Finally, researchers working at UCLA School of Dentistry are working on discovering and validating biomarkers for periodontal disease in saliva. The biomarkers will then be detected by the Oral Fluid NanoSensor Test (OFNASET) that is able to detect multiplex protein and transcriptomic biomarkers simultaneously. The team at UCLA have developed a OFNASET that provides low cost, real time, highly sensitive and specific POC technology optimized to saliva for clinical applications (Fig. 1).117

Figure 1: The Oral Fluid NanoSensor Test (OFNASET) technology platform is a point-of-care technology optimized for ultra-sensitive and specific detection of salivary biomarkers for disease detection.
figure 1

In addition it is multiplexible (proteins and nucleic acids RNA/DNA), robust and inexpensive.

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Implications for dental care practitioners

As patients with periodontal disease come from a wide range of socioeconomic backgrounds, disparities in accessibility to professional treatment are present.118 Even for those with the ability to afford treatment, it is not confirmed that the minor clinical attachment gain justifies the higher cost and extended recovery time of surgical interventions.29,119,120 POC periodontal disease testing, whether used in a dental office, health clinic, or purchased directly by the consumer to be used at home would allow healthcare providers to more closely monitor disease progression and allow more time for them to advise their patients of periodontal self-care before the disease becomes irreversible.121 Utilising Using a non-invasive, real-time diagnostic tool would allow quicker diagnostic capabilities for practitioners and increased access to dental care in efforts to reduce disparities.95

Future directions

A most important translational goal for salivary biomarkers to achieve clinical reality is the ability to definitively and pivotally validate salivary biomarkers at the regulatory level (Food and Drug Administration). The opportunity to utilise saliva as a diagnostic alternative to blood and urine has been a long sought-out goal that is just recently moving into chairside availability due to advances in robust scientific platform and biomarker validations. However, the potential to utilise saliva for diagnostic screenings for oral and systemic diseases simultaneously remains unfulfiled. It would be ideal to be able to assay a single saliva sample for a wide range of diseases using genomic, proteomic, and bacterial markers. Currently, many salivary biomarkers can test for a number of different diseases, but for saliva to live up to clinical expectations the technology should be able to detect many different biomarkers from a range of diseases simultaneously.122 Numerous research groups are working toward this goal by combining microbial markers from periodontal pathogens with salivary biomarkers from host-response changes to those pathogens to further expand the clinical viability of salivary diagnostics.42,91 As saliva is in constant contact with the periodontium, it is an advantageous diagnostic fluid for periodontal disease because the analytes reflect the current disease activity that will allow dentists to appropriately determine the severity and guide treatment options.95 Various diagnostic tests are commercially available, and the development of point-of-care tests is expanding. Salivary diagnostics for periodontal disease is promising and could facilitate the diagnostics and treatment in a clinical practice by dental practitioners in a near future.

Disclosures

David Wong is co-founder of RNAmeTRIX Inc., a molecular diagnostic company. He holds equity in RNAmeTRIX, and serves as a company Director and Scientific Advisor. The University of California also holds equity in RNAmeTRIX. Intellectual property that David Wong invented and which was patented by the University of California has been licenced to RNAmeTRIX.