Introduction
The most common chronic inflammatory conditions worldwide and collectively the most common diseases known to mankind, are inflammatory periodontal diseases.1 Periodontal diseases include gingivitis, where the inflammation confined to the gingiva is reversible with good oral hygiene, and periodontitis, an extension of the inflammation that results in tissue destruction and alveolar bone resorption.1 Periodontitis is very common with 10-15% of adults being afflicted with severe periodontitis and 40-60% being affected by moderate periodontitis.2 Several forms of periodontitis have been recognized; however, the predominant category is chronic periodontitis (CP) which remains the number one cause of tooth loss in adults worldwide.3 The goal of periodontal diagnostic tools and procedures is to provide useful information to the clinician on the periodontal disease type, location, and severity. This information will serve as the basis for treatment planning and monitoring of disease.4 Traditional periodontal clinical diagnostic parameters include probing depths, bleeding on probing, clinical attachment levels, plaque index, and radiographs.5 The strengths associated with these traditional tools are that they are easy to use, cost-effective, and are relatively non-invasive.5 However, these traditional diagnostic procedures are limited in that only disease history, not current disease status and activity, can be assessed and identified.5,6 For example, clinical attachment loss readings by the periodontal probe and radiographic evaluation of alveolar bone loss measure damage from past episodes of destruction and require a 2 to 3 mm threshold change before a site can be identified as having experienced a significant anatomic event.5 Even in instances when patients’ treatments are monitored over time it can be difficult to use these clinical parameters to make a definitive periodontal diagnosis.5 As another example, does a patient who has been treated with non-surgical periodontal treatment and now has several sites with residual probing depths that bleed on probing still have periodontitis that requires further active therapy or surgical treatment, or is the condition stable and the disease in remission?7 Moreover, other limitations such as the difficulty in precisely duplicating the insertion force, probe placement and angulation exist.8 Radiographs, a key factor in determining the severity of periodontitis and bone-related damage, have limited sensitivity and only reveal change in bone after 30% to 50% of bone loss has occurred.8 Furthermore, radiographs cannot be taken at each visit due to excess radiation exposure to the patient.8 Advances in oral and periodontal disease diagnostic research are consequently moving forward toward methods whereby periodontal diagnosis and risk can be identified and quantified by measures that are objective7, minimally invasive, less technique sensitive, less time consuming and that are able to identify active and potential periodontal disease. New developments in periodontal diagnostic research will be discussed below.
Figure 1
Three patients using test strips. The first row of test strips represents the baseline and the second row are the test strips where the oral rinse was administered. Patient 1 shows a positive test result, whereas patients 2 and 3 are negative.
Saliva
Saliva as a diagnostic tool for periodontal disease has been the subject of considerable research and the proposed markers for disease include proteins of host origin (i.e. enzymes, immunoglobulins), phenotypic markers (epithelial keratins), host cells, hormones (cortisol), bacteria and bacterial products, volatile compounds and ions.6 In comparison to healthy subjects, patients with periodontitis have higher concentrations of host-derived salivary enzymes due to contributions of Polymorphonuclear Leukocytes (PMNs), bacteria and the presence of connective tissue destruction seen in association with periodontal disease.6 PMNs-derived enzyme activity, characteristic of acute inflammation, specifically collagenase, elastase, and gelatinase, as well as bacterial enzymes, are higher in patients with periodontitis.6 These enzymes are found to be highest before treatment and decrease following periodontal treatment and therapy.6 Since PMNs contribute to tissue destruction and to the salivary enzyme pool, evaluation of the number of PMNs in saliva appears to be one way to diagnose the presence of periodontal disease in humans; however, the practicality of doing this was questionable6 until 1978 when Raeste & Aura first proposed the idea of using PMN quantification employing a rinse to assess periodontal disease.9 It was found that leukocyte counts between healthy and periodontally diseased subjects were highly significant and therefore the rinse had value on its own for testing for periodontal disease severity.9 Bender, Thang, & Glogauer, 2006, quantified oral PMN levels using a hemocytometer in a technique coined oral rinse assay.9 Thang et. al, 2006, concluded that the oral rinse assay is a valid, reproducible and effective way of collecting and quantifying oral PMN levels and this technique can be used to assess the presence and/or severity of periodontal disease.9 A modification of this technique is the colourimetric assay, where ABTS (2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid, a colour changing redox agent) was added to the oral rinse samples.10 Landzberg, 2009, correlated colour changes in the reagent solution and periodontal disease, providing a simpler method of measuring oral PMNs.10 A further modification of the colourimetric assay using the same reagents but observing a colour change on a test strip has been developed by Dr. Michael Glogauer. This technique is essentially the same as the colourimetric assay except the colour change is observed on the test strips rather than within the rinse samples collected (Fig. 1).
Oral Plaque
Although the pathogenesis of periodontitis is multifactorial, the development of periodontitis is modulated by microbial biofilm eliciting an inflammatory host reaction and thus the etiology of periodontitis is polymicrobial.3 In particular, the proliferation of Gram-negative anaerobic species such as Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia has been linked to the disease progression of periodontitis.3 Historically, there has been a focus on a small sub-set of CP (chronic periodontitis) associated microorganisms in the gingival sulcus; this means overlooking the impact of potentially large numbers of other oral bacterial species in the infectious process.3 There is emerging evidence, using DNA sequencing, that suggests that periodontal destruction is associated with many uncultivable and uncharacterized disease indicator organisms (Fig. 2).3 A more complete characterization of bacterial biomarkers of CP will lead to the development of new therapeutics, improved diagnostics, and alternative methods to monitor periodontal treatment success.3 Galimanas et al., 2014 demonstrated that the tongue dorsum and supragingival sites can be used as alternative sample sites for the detection and enumeration of bacterial biomarkers associated with CP (Fig. 2.).3 As an easily accessible body site, the tongue dorsum for plaque collection, is convenient and less invasive.3
Infrared (IR) Spectra of Gingival Crevicular Fluid (GCF)
GCF is defined as either serum transudate or inflammatory exudate composed of serum and locally generated materials such as tissue-breakdown products, inflammatory mediators, and antibodies formed against dental plaque bacteria.8 Production of GCF is controlled by the passage of fluid from capillaries into tissues and the removal of this fluid by lymphatics.8 The amount of production of GCF at a given site significantly increases with the severity of gingival inflammation clinically and histologically.8 Over 65 biologically active components have been identified in GCF providing many diagnostic and prognostic markers for periodontitis; these include: 1) the presence of specific bacteria (including Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans previously known as Actinobacillus actinomycetemcomitans, 2) bacterial products of metabolism (i.e., volatile sulfur compounds or specific proteases), 3) biomarkers involved in the disease process but produced by the host (i.e., matrix metalloproteinases, neutrophil elastase, and alkaline phosphatase), 4) biomarkers of tissue damage (i.e., hydroxyproline/collagen fragments), and 5) other markers of the inflammatory process, such as prostaglandin E2 and interleukin- 1.8 However, such potential biomarkers are generally studied individually or, rarely, in small numbers possibly explaining why the predictive value of potential biomarkers has proved insufficient for effective routine clinical use.8
Unlike traditional biochemical analyses, IR (infrared) analysis of GCF measures the total content of GCF thereby proving to be a more powerful diagnostic and prognostic tool for periodontal diseases.8 Xiang et al., 2009 demonstrated that when using IR spectroscopy to characterize GCF from healthy, gingivitis and periodontitis sites, specific spectral signatures clearly demarcated healthy and diseased tissues.8 They demonstrated that subtle differences in spectral band intensity and positions arising from three major components – lipid, protein, and DNA, – were observed in GCF from healthy, gingivitis, and periodontitis groups.8 It was found that compared to healthy subjects, the DNA content in the inflammatory GCF was higher in diseased subjects, suggesting an early phase during the inflammatory process with active enrolled leukocytes, bacteria, and shedding epithelial cells.8 Moreover, they found increased protein and lipid signals at diseased sites.8 Another advantage to IR spectroscopy other than its ability to capture the composite molecular contents of GCF, is its ability to provide a qualitative diagnosis of periodontal inflammatory status.8 This can be achieved by using statistical analysis to correlated, observed spectral differences of GCF from inflammatory conditions (gingivitis and periodontitis) and normal healthy status.8 The accuracy of diagnosing periodontitis this way was found to be 98.4% for the training set and 93.1% for the validation set.8 Other advantages of IR spectroscopy of GCF for the screening and diagnosis of periodontitis are as follows: reagent free, small sample volumes, unprocessed GCF samples, the process is readily automated, IR spectroscopy is straightforward requiring minimal spectroscopy training of operators, and GCF samples are easily collected.8
Near-infrared (NIR) Spectroscopy
Near-infrared (NIR) spectroscopy, a type of optical spectroscopy, can be used to monitor hemodynamic and edema-based markers of oral cavity soft tissues.4,8 Within visible and NIR spectral regions, several light-absorption bands reflect key inflammatory events.8 For example, the wavelength region 500 to 600 nm is dominated by the absorption from oxygenated hemoglobin and deoxygenated hemoglobin in the capillary bed gingival tissue.8 Therefore, NIR spectroscopy can assess the balance between tissue oxygen delivery and oxygen use.8 Moreover, tissue edema, another index used as a marker of gingival inflammation, can be measured by NIR spectroscopy.8 Periodontal edema ensues from an increase in vascular permeability in response to bacterial etiology leading to interstitial fluid accumulation subsequently leading to the release of a variety of inflammatory exudates in the gingival crevice.8 Optical spectroscopy, in other words, is a non-invasive method of assessing the balance between tissue oxygen delivery and oxygen use and hemoglobin-oxygen saturation of tissues and the degree of tissue perfusion as well as a measure of tissue edema.4,8 Xiang et al., 2009 demonstrated, using NIR spectroscopy, that tissue oxygenation at periodontitis sites was significantly decreased (P <0.05) compared to gingivitis and healthy controls (Fig. 3).8 Decreased oxygen saturation reflects tissue hypoxia as a response to ongoing inflammatory response leading to increased oxygen consumption.8 Moreover, since anaerobic bacteria predominantly colonize periodontal pockets in destructive periodontal disease, diminished oxygen tension in deep pockets would therefore be expected to further promote the growth of these anaerobic bacteria.8 Xiang et al., 2009 also found high tissue water content at the site of the periodontitis compared to the healthy sites causing periodontal edema (Fig. 3). Optical spectroscopy in the application of periodontal inflammation and periodontal disease diagnosis is an attractive technology because spectra can be captured instantaneously, no consumables need to purchased or developed, and once the equipment is in place, it is very inexpensive and minimal training is required.8
Optical Coherence Tomography (OCT) for Periodontal Diagnosis
OCT is an imaging technique in which a lightly focused light beam is scanned across the tissue surface of interest producing a high-resolution cross-sectional images of the biologic structures.8 OCT imaging can provide three-dimensional imaging of periodontal soft tissues and bone at a high resolution (Fig. 4) thereby offering the potential for identifying active periodontitis before significant alveolar bone loss occurs.8 Therefore, because the optical nature of OCT does not require direct contact with the tissue, thus not compressing the soft tissue, OCT may prove to be a more reproducible and reliable method of determining the attachment level than traditional probing methods.8
The Ultrasonographic Probe
The ultrasonographic probe uses ultrasound waves to detect, image and map the upper boundary of the periodontal ligaments, and its variation over time, as a way to detect the presence of periodontal disease.4 The ultrasonographic probe has a removable tip, space for a 2 mm active area transducer, a water line input that runs through the probe handle and empties into a small open area around the transducer, and an electronics input-output cable that also runs through the base and is connected to the transducer.4 Using a stream of water, a narrow, high-frequency ultrasonic pulse is projected into the gingival sulcus/periodontal pocket.4 The waves emitted from the ultrasonic interact with the periodontal tissue and the echoes carry relevant information back to the transducer.4 Because of its ability to detect smaller increments of anatomic change, the ultrasonographic probe can potentially detect early tissue breakdown and additional histologic information such as that of tissue thickness and inflammation.4 Therefore, the ultrasonographic probe is a non-invasive, painless, and less prone to examiner variability, potentially more sensitive, and may yield additional histological information when compared to the traditional manual periodontal probing.4
Conclusion
Given the complexity of periodontal disease, it is unlikely that one single clinical or laboratory test or examination can address all the issues that surround diagnosis and prognosis. Although these new minimally invasive methods seem promising, they are best used as complementary to one another and employed with conventional clinical and radiographic examinations. These techniques require more scientific research to be considered more accurate, reliable, and stand-alone methods in the diagnosis and prognosis of periodontal disease.OH
Oral Health welcomes this original article.
References
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- Huda S. Assay of Oral Polymorphonuclear Neutrophils for Assessment of Oral Inflammation in Pregnant Women (Unpublished master’s thesis). University of Toronto Faculty of Dentistry; 2012.
- Landzberg M. Evaluation of Colorimetric Assay As A Screening Test For Periodontal Disease (Unpublished master’s thesis). University of Toronto Faculty of Dentistry; 2009.
Dr. Zahra Dorna Mojdami is a graduate of the University of Western Ontario where she obtained her DDS in 2012. Currently, she is pursuing her specialty and MSc. degree in Dental Public Health at the University of Toronto, Faculty of Dentistry and also works as a general dentist in private practice in Toronto.
Dr. Michael Glogauer DDS PhD Dip Perio is a Professor at the University of Toronto. His research and clinical interests focus on the role of the oral innate immune system in maintenance of health. He is currently focusing on using oral innate immune biomarkers to detect early stages of periodontal diseases through his role as Scientific Director at the Mt. Sinai Hospital’s Centre for Advanced Dental Research and Care. He is a periodontist at OMGPerio.ca.
Dr. Amir Azarpazhooh received his dental degree from Mashhad, Iran (2001), his specialty training in Canada at the University of Toronto in Dental Public Health (2007) and Endodontics (2010), followed by his PhD (2011). He is an Associate Professor in the Faculty of Dentistry, in Dental Public Health and Endodontics, with a cross appointment to the Clinical Epidemiology Program of the Institute of Health Policy, Management and Evaluation of the Faculty of Medicine, and the Toronto Health Economics and Technology Assessment (THETA) Collaborative of the University of Toronto. He is also the Head of Divisions of Endodontics and Research at the Department of Dentistry, Mount Sinai Hospital, Toronto and a Clinician Scientist with the Lunenfeld-Tanenbaum Research Institute of Mount Sinai Hospital. As a dual specialist, his area of research crosses between Dental Public Health and clinical disciplines, and includes knowledge synthesis and implementation, epidemiological research in dentistry, investigating the link between oral and general health, and shared decision making in clinical dentistry. Currently, his attributed grants and awards total more than CDN $1.25M and these have enabled him to manage a strong group of graduate students over the years. To date, he has published six book chapters, and more than 150 papers, abstracts and reports and has presented at over 50 national and international scientific meetings. Amir is also a practicing endodontist, with part-time private practice in Toronto as well as a hospital practice in Mount Sinai Hospital providing endodontic care to patients when their medical, physical or mental status indicates the need for a hospital environment.
