Written by John T. Grbic, DMD, MS, MMSc, and Steven P. Engebretson, DMD, MS Wednesday, 30 April 2003 19:00
Traditionally, periodontal disease has been diagnosed by a radiographic and clinical examination including the assessment of plaque, gingival inflammation (usually bleeding on probing), probing depth, and attachment level. A major drawback to a traditional periodontal evaluation is its inability to determine which patients are stable and which patients have “active” disease.
In the late 1970s and early 1980s, landmark studies by Socransky and colleagues1,2 changed the prevailing concept of periodontal disease from being a slow, continuously progressing disease to one where periodontal attachment loss occurs in random bursts with periods of exacerbation and quiescence. During these “active” episodes of periodontal disease, significant amounts (typically greater than 2 mm) of periodontal attachment were lost at a specific site over a short period of time. Furthermore, they reported that clinical parameters such as probing depth and bleeding on probing were limited in their ability to predict ”active” periodontal disease.3 Other studies4,5 demonstrated that an individual’s risk for periodontal disease varied significantly, with a majority of active sites clustered in a small percentage of patients. The need to determine which patients are at risk for “active” progressive periodontal disease led to an increased interest in the development of new, improved methods of periodontal diagnosis.
The development of new, nontraditional periodontal diagnostic tests is related to our improved understanding of the pathogenesis of periodontal disease and has led to the development of assays that accurately measure the subgingival microbial challenge and the host response to this challenge. This concept of evaluating a disease with laboratory tests was relatively new in dentistry, but has been used extensively in medicine.
The goal for researchers has been to develop the ideal test for periodontal disease that would be able to (1) predict which patients will experience attachment loss (“active” periodontal disease) in the near future, and (2) predict which teeth or which sites will experience attachment loss in the near future. In addition to predicting which patients or sites are at higher risk of becoming active, diagnostic tests can be used to categorize patients into different disease categories; ie, aggressive or chronic periodontitis, or response or no response to treatment. They can also be used to determine the prognoses of either the entire patient or a specific tooth. In addition, an ideal periodontal diagnostic test would have to be economically feasible and easy to use in a dental office setting.
INTERPRETATION OF RESULTS FROM DIAGNOSTIC TESTS
|Figure 1. Initial concepts of periodontal disease diagnostic testing: testing for the risk of periodontal disease progression.|
Figure 1 illustrates a typical study design used to determine whether the presence of a specific marker is related to periodontal attachment loss. Ideally, you would like to demonstrate a change in marker being evaluated immediately prior to the patient experiencing periodontal attachment loss6. Outcomes from these trials are presented either as the sensitivity and specificity of the test or as a measure of risk.7 A clinician evaluating new laboratory-based diagnostic methods must understand the differences between these 2 methods of evaluating the utility of a diagnostic test.
Sensitivity refers to the ability of a diagnostic test to detect the presence of the disease. For example, a sensitivity of 80% on a test implies that 80% of the individuals with a disease were detected by this test; 20% of the patients with the disease were not. Specificity refers to the ability of a diagnostic test to detect the absence of a disease. Therefore, a specificity of 90% suggests that 90% of the individuals without the disease gave a negative result with the test while 10% without the disease were positive for the test. Sensitivity and specificity tend to be inversely related, and the relationship between the 2 is determined by the criteria used to determine a positive test. The optimal balance between sensitivity and specificity is determined by plotting a curve comparing sensitivity and specificity (known as the “receiver-operator” curve).
Recently, the use of risk assessment has become popular for evaluating diagnostic tests. Risk assessment has several advantages over the use of sensitivity/specificity. It helps inform the practitioner of the probability of developing a disease based on a specific diagnostic finding. The data is usually presented as odds ratios that correspond to the probability of developing a disease compared to a specified control group. An example of a common test that uses risk is the test for serum cholesterol. Increasing serum cholesterol levels directly correspond to increasing levels of risk of a cardiovascular event (myocardial infarction or stroke), and thus provide both the practitioner and the patient with a more easily understood quantitative assessment of the probability of developing a disease. In periodontal diagnostic testing, risk assessment is used to determine the probability of having active periodontal disease in the near future based on a specific laboratory or clinical finding.
DIAGNOSTIC TESTING FOR PERIODONTAL DISEASE
The search for new diagnostic technologies in periodontal disease has predominately focused on evaluating the microbial challenge in the periodontium or the host response to this infection.
Considerable interest has focused on developing diagnostic tests based on the presence or absence of specific subgingival bacteria. Various assays have been used to detect the presence of specific periodontal pathogens, including culture techniques, immunoflourescence, and DNA probe technology.8 Using culture techniques requires that a viable bacterial sample be obtained from the patient and transferred to the laboratory. Because of the need to preserve anaerobic conditions, the sampling techniques are more technically demanding, making this methodology less useful than other techniques that do not require viable bacteria. However, culture and sensitivity testing are useful in determining antibiotic treatment options for patients.
The most commonly used bacteriologic test for periodontal pathogens is based on DNA probe technology. This technology has several advantages over culture techniques, including increased sensitivity8 (approximately 103 bacteria can be detected) and elimination of the need for viable bacteria (since nonvital bacteria can be detected using this technology). The sampling technique for these assays is relatively noninvasive, using either endodontic paper points or a sterile curette to obtain bacteria from the subgingival environment. A number of bacteria have been studied for their relationship to “active” periodontal disease. The strongest evidence of an association with periodontal disease progression are the organisms found in Haffajee and Socransky’s “red complex.”9 These include Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola. In addition to these bacteria, Actinobacillus actinomycetemcomitans has been found to be associated with aggressive periodontitis. Although the above-mentioned bacteria have been associated with an increased risk of periodontal disease progression, they have been found in significant numbers in patients with nonprogressing periodontal disease.10
Another important methodologic issue for the application of microbial testing is which sites should be sampled and are these sites representative of the entire dentition? Because sampling every site within a patient’s mouth for bacteria would be impractical and economically prohibitive, specific sites must be chosen. Most clinicians tend to sample the most diseased sites within a patient’s mouth and use the results to classify the entire patient as being at risk for periodontal disease progression. Clearly, the possibility exists that the collected sample will not be representative because of the limitations of sampling.11
HOST RESPONSE TO PERIODONTAL DISEASE
Studies in the past 25 years have brought about a significant improvement in our understanding of the response of the host to the microbial challenge in the periodontium. The improved understanding of the inflammatory and immune responses in periodontal disease has led to the development of assays that identify host factors responsible for periodontal disease progression.12 Analysis of the host response for diagnostic purposes has involved the quantification of specific host-derived molecules within gingival crevicular fluid, serum, or saliva.13
Gingival Crevicular Fluid Assays
Gingival crevicular fluid (GCF) is a serum transudate or an inflammatory exudate found in the gingival sulcus.13 GCF originates primarily from the blood vessels surrounding the junctional and sulcular epithelium. As this fluid travels through the surrounding gingival connective tissue and eventually through the junctional epithelium, it transports molecules found in these tissues into the gingival sulcus. In addition, significant numbers of polymorphonuclear leukocytes and epithelial cells as well as bacterial cells can be found in GCF.13 GCF can be collected noninvasively by inserting a methylcellulose filter paper strip into the gingival sulcus. These GCF strips absorb the fluid from the gingival sulcus and are removed usually after a specific time period. However, although not technically difficult, care must be taken while collecting GCF to prevent contamination of the GCF strip with blood and saliva. The volume of fluid collected can be determined by using an instrument that measures the change in electrical capacitance, know as a Periotron, and typically ranges from less than 0.1 to 2.0 microliters of fluid. Components of GCF are eluted off the strip and then analyzed by either an immunologically based assay (enzyme-linked immunoabsorbence assay or ELISA) or by an enzyme-substrate assay.13 These highly sensitive assays are able to quantify the presence of specific molecules in the GCF.
Because of the advent of GCF analysis, hundreds of molecules have been analyzed in GCF, although only a relatively small number of them have shown a consistent association with periodontal disease activity and have a potential as diagnostic tests.13 Briefly, we will review some of the molecules that have demonstrated significant association with periodontal disease.
Markers of Cell Death
Aspartate aminotransferase (AST) is an enzyme found in the cytoplasm of cells and is used as a measure of cell death. Upon cell death and disruption of the cell membrane, AST leaks from the cytoplasm into the interstitial fluid.14 The concept underlying the use of AST as a diagnostic test assumes that during phases of periodontal destruction, large numbers of cells will die, releasing AST into the GCF. Chambers et al15 reported that elevated levels of AST were associated with a 9 to 16 times greater risk of experiencing active periodontal tissue destruction. Other markers of cell death, such as lactate dehydrogenase (LDH), were not found to be associated with periodontal disease progression.16
MARKERS OF THE ACUTE INFLAMMATORY RESPONSE
Both ß-glucuronidase and elastase are enzymes found in the lysosomal granules of polymorphonuclear leukocytes (PMNs). Levels of these enzymes are related to the degree of acute inflammation present in the periodontium and gingival sulcus.17 Lamster et al,18 in a multi-center trial, evaluated whether GCF levels of ß-glucuronidase could predict attachment loss in the following 3 months. They found that subjects with elevated levels of GCF ß-glucuronidase were 6 to 14 times more likely to experience significant clinical attachment loss in the next 3 months than patients without elevated levels of ß-glucuronidase. Armitage et al19 found that elastase was also elevated in patients with active periodontal disease.
Both of these assays can be performed rapidly in a dental office setting, thus providing immediate feedback to the dentist and patients.17 Matrix metalloproteinases (MMPs) released by inflammatory cells have also been shown to aid in the diagnosis of periodontal disease.20 Azmak et al21 evaluated the use of a chairside MMP-8 GCF dipstick test and found it very effective in evaluating the outcome of periodontal treatment.
Cytokines are molecules that modulate the function of a wide variety of cells and are involved in regulating the immune and inflammatory response. Although a large number of cytokines have been studied, the cytokine with the most promise for diagnostic testing is known as interleukin-1ß. (IL-1ß).22,23 IL-1ß is produced by a wide variety of cell types, but the primary producer of IL-1ß in the gingival tissues is the macrophage. IL-1ß has a number of biologic effects, including initiating the acute phase response to infection. However, in the periodontium, it likely functions to mediate connective tissue destruction and osteoclastic bone resorption.24 GCF levels of IL-1ß have been found to be consistently associated with the severity of periodontal disease.22 However, the use of IL-1ß as an in-office diagnostic is limited because analysis requires sophisticated laboratory equipment and expertise.
Immunoglobulins are produced in periodontal tissues and immunoglobulins are present in the GCF.25 Levels of immunoglobulin G (IgG) have been found not to have a consistent association with periodontal disease, although subtypes of IgG may be found in subjects at higher risk for periodontal disease progression.26 Interestingly, immunoglobulin A (IgA) has been shown to be elevated in healthy subjects and subjects who have a decreased rate of periodontal disease progression, suggesting that it may be a marker of protection.27,28
In summary, GCF analysis has demonstrated its utility as predictor of risk for periodontal attachment loss. However, GCF has similar limitations as microbial testing; namely, which sites should be sampled?
Serum Diagnostics for Periodontal Disease
Serum can be used to measure the levels of antibodies to specific periodontal pathogens. Several investigators29,30 have found that serum antibodies to periodontal pathogens are generally elevated in patients with periodontal disease, although there is a group of periodontitis patients who fail to respond with an appropriate antibody response.31 Specific serum antibody has been used to differentiate patients as having either aggressive or chronic periodontitis.29
Salivary Diagnostics for Periodontal Disease
Saliva is a fluid with a vast and underutilized potential as a diagnostic not only for periodontal disease, but systemic diseases as well.32 Whole saliva contains the saliva from the salivary glands as well as the outflow from gingival sulci (GCF). The use of whole saliva provides a measure of the entire patient, thus avoiding the sampling problems associated with collecting microbial samples and GCF. A recent study by Lamster et al,33 demonstrated the ability of ß-glucuronidase in saliva to differentiate patients by levels of periodontal probing depths and gingival inflammation. Although preliminary studies have been promising, no long-term studies evaluating the use of a marker for periodontal disease progression in saliva have been performed.
CURRENT STATUS OF PERIODONTAL DIAGNOSTIC TESTING
Despite the development of several assays that measure the risk of periodontal disease progression, periodontal diagnostics have not been commercially successfully. Lamster and Pope34 examined the issues involved by evaluating the technical, regulatory, business, and marketing issues surrounding the development of a diagnostic test for periodontal disease. They found that the technical and regulatory issues involved with periodontal diagnostic testing were relatively minor. However, they found that business and marketing concerns have prevented the introduction of periodontal diagnostics to the dental marketplace. These business and marketing issues included: (1) the lack of reimbursement for diagnostic tests by insurance companies; (2) the high costs for pharmaceutical or biotechnology companies to develop and gain approval of a periodontal diagnostic through the Food and Drug Administration; and (3) the lack of acceptance of periodontal diagnostic testing by practitioners.
They concluded that for a periodontal diagnostic test to be successful, the practitioner must have a clear understanding of the relationship between periodontal diagnostic testing and treatment as well as an appreciation of the financial and health benefits of diagnostic testing. This would require a “cultural” change for dentists who are not trained to use diagnostic testing in their practices.
WHEN TO USE DIAGNOSTIC TESTING
|Figure 2. The future of periodontal disease diagnostic testing: testing for the risk of systemic diseases and therapeutic endpoints.|
An important question concerning diagnostic testing is when should tests be performed. Originally, periodontal diagnostic tests were designed for use prior to the initiation of periodontal therapy (Figure 2). A thorough clinical examination and general health history provide a great deal of information for the clinician regarding who is at risk for future periodontal attachment loss. Testing at this time may provide baseline values to assess changes because of treatment.
In addition to being used to diagnose a disease, diagnostic testing can determine when a chronic, persistent disease has been adequately treated (establishment of a therapeutic endpoint). An example of this in medicine is evaluation of Helicobacter pylori infection, which leads to stomach ulcers in susceptible patients. A physician treating H. pylori will typically monitor antibody levels to H. pylori to determine whether the infection has been appropriately treated.35 If initial therapy fails to achieve elimination of infection, treatment may be necessary. Similarly, periodontal diagnostic testing can be used to determine when a patient has received adequate therapy. Currently, the endpoint of periodontal therapy is based on the reduction of probing depth and gingival inflammation. However, more sensitive measures of periodontal infection exist, including analysis of post-treatment levels of periodontal pathogens as well as a reduction in the host response to these pathogens. For example, patients who persist with high levels of periodontal pathogens or elevated levels of inflammatory mediators in their saliva can be treated with additional therapy, including antibiotic therapy. However, to appropriately use periodontal diagnostic testing to monitor the therapeutic outcome of a patient, pretreatment diagnostic testing should be performed. The use of periodontal diagnostic tests to determine the conclusion of the “active” phase may help reduce the rate of refractory disease.
A persistent problem for clinicians remains the decision when to provide additional therapy to a patient who has been receiving periodontal maintenance therapy. Periodontal diagnostic testing can be used in these patients to determine when further therapy is necessary as well as to help establish an appropriate recall interval for each patient.
THE FUTURE OF PERIODONTAL DIAGNOSTICS
|Figure 3. A typical study design for determining the ability of a diagnostic test to predict clinical attachment loss.|
Recently, a number of studies have demonstrated a relationship between periodontal disease and systemic diseases, including cardiovascular disease.36 The mechanism by which periodontal infection may contribute to cardiovascular disease is not resolved, but likely relates to the periodontal infection and local inflammatory response affecting systemic inflammation. This increased systemic inflammation would lead to increased artheroma formation and increased potential for thrombotic events. However, it has become apparent that not all patients with periodontal disease are at high risk for the development of cardiovascular disease. Furthermore, periodontitis patients who are at high risk for the development of cardiovascular disease cannot be identified by a clinical examination. Therefore, a potential future use of periodontal diagnostic testing is to determine which patients with periodontal disease may be at risk for increased occurrence of systemic disease (Figure 3). A molecule which has demonstrated promise as a potential risk marker in oral fluids is IL-1ß, which plays an important role in initiating inflammatory and immune responses, both locally in the periodontium and systemically. Beck and Offenbacher36 reported that GCF levels of IL-1ß were significantly associated with the thickness of carotid arterial walls as measured by ultrasound in a population of 6,000 adults. This finding suggests that biochemical measures of periodontal inflammation may be related to the development of cardiovascular disease. Further studies are necessary to define if other markers in oral fluids or the presence of specific periodontal pathogens can be associated with the risk for cardiovascular disease.
Although periodontal diagnostic testing was initially developed to determine which patients were at risk for active periodontal disease, the future of periodontal diagnostic testing may ultimately be in the area of “periodontal medicine”; specifically to determine which patients are at risk for developing systemic disease and when has that risk been adequately reduced as a result of effective treatment.
1. Socransky SS, Haffajee AD, Goodson JM, et al. New concepts of destructive periodontal disease. J Clin Periodontol. 1984;11:21-32.
2. Socransky SS, Haffajee AD. The nature of periodontal diseases. Ann Periodontol. 1997;2:3-10.
3. Haffajee AD, Socransky SS, Goodson JM. Clinical parameters as predictors of destructive periodontal disease activity. J Clin Periodontol. 1983;10:257-265.
4. Haffajee Ad, Socransky SS, Lindhe J, et al. Clinical risk indicators for periodontal attachment loss. J Clin Periodontol. 1991;18:117-125.
5. Grbic JT, Lamster, IB . Celenti RS, et al. Risk indicators for future attachment loss in adult periodontitis. Patient variables. J Periodontol. 1991;62:322-329.
6. Lamster IB. Evaluation of components of gingival crevicular fluid as diagnostic tests. Ann Periodontol. 1997;2:123-137.
7. Greenstein G, Lamster I. Understanding diagnostic testing and risk assessment for periodontal diseases J Periodontol. 1995:66:659-666.
8. Zambon JJ, Haraszthy VI, The laboratory diagnosis of periodontal infections. Periodontol 2000. 1995;7:69-82.
9. Socransky SS, Haffajee AD. Dental Biofilms: diffcult therapeutic targets. Periodontol 2000. 2002;28:12-55.
10. Zambon JJ. Principles of evaluation of the diagnostic value of subgingival bacteria. Ann Periodontol. 1997;2:138-148.
11. Haffajee AD, Socransky SS. Effect of sampling strategy on the false-negative rate for detection of selected subgingival species. Oral Microbiol Immunol. 1992;7:57-59.
12. Lamster IB, Celenti RS, Jans HH, et al. Current status of tests for periodontal disease. Adv Dent Res. 1993;7:182-190.
13. Lamster IB, Grbic JT. Diagnosis of periodontal diseae based on analysis of the host response. Periodontol 2000. 1995;7:83-99.
14. Persson GR, DeRouen TA, Page RC. Relationship between gingival crevicular fluid levels of aspartate aminotransferase and active tissue destruction in treated chronic periodontits patients. J Periodont Res. 1990;25:81-87.
15. Chambers DA, Imrey PB, Cohen RL, et al. A longitudinal study of aspartate aminotransferase in human gingival fluid. J Periodontal Res. 1991;26:65-74.
16. Lamster IB, Oshrain RL, Harper DS, et al. Enzyme activity in crevicular fluid for detection and prediction of clinical attachment loss in patients wirth chronic adult periodontitis: 6 month results. J Periodontol. 1988;59:516-523.
17. Lamster IB. In-office diagnostic tests and their role in supportive periodontal treatment. Periodontol 2000. 1996;12:49-55.
18. Lamster IB, Holmes LG, Gross KB, et al. The relationship of b-glucuronidase activity in crevicular fluid to probing attachment loss in patients with adult periodontitis: findings from a multi-center study, J Clin Periodontol. 1995;22:36-44.
19. Armitage GC, Jeffcoat MK, Chadwick DE, et al. Longitudinal evaluation of elastase as a marker for the progression of periodontitis. J Periodontol. 1994:5:120-128.
20. Embery G, Waddington RJ, Hall RC, et al. Connective tissue elements as diagnostic aids in periodontology. Periodontol 2000. 2000;24:193-214.
21. Azmak N, Atilla G, Luoto H, et al. The effect of subgingival controlled-release delivery of chlorhexidine chip on clinical parameters and matrix metalloproteinase-8 levels in gingival crevicular fluid. J Periodontol. 2002;73:608-615.
22. Engebretson SP, Grbic JT, Singer R, et al. GCF IL-1beta profiles in periodontal disease. J Clin Periodontol. 2002;29:48-53.
23. Stashenko P, Fujiyoshi P, Obernesser MS, et al. Levels of interleukin 1ß in tissue from sites of active periodontal disease. J Clin Periodontol. 1991;18:548-554.
24. Takahashi K, Mooney J, Frandsen EV, et al. IgG and IgA mRNA-bearing plasma cells in periodontitis gingival tissue and immunoglobulin levels in gingival crevicular fluid. Clin Exp Immunol. 1997;107:158-165.
25. Rheinhardt RA, McDonald TL, Bolton RW, et al. IgG subclasses in gingival crevicular fluid from active versus stable periodontal sites. J Periodontol. 1989;60:44-50.
26. Grbic JT, Singer RE, Jans HH, et al. Immunoglobulin isotypes in gingival crevicular fluid: possible protective role of IgA. J Periodontol. 1995;66:55-61.
27. Grbic JT, Lamster IB, Fine JB, et al. Changes in gingival crevicular fluid levels of immunoglobulin A following therapy: association with attachment loss. J Periodontol. 1999;70:1221-1227.
28. Ishikawa I, Nakashima K, Koseki T, et al. Induction of the immune response to periodontopathic bacteria and its role in the pathogenesis of periodontits. Periodontol 2000. 1997;14:79-111.
29. Ebersole JL. Systemic humoral immune responses in periodontal disease. Crit Rev Oral Biol Med. 1990;1:283-331.
30. Ebersole JL, Taubman MA. The protective nature of host responses to periodontal diseases. Periodontol 2000. 1994;5:112-141.
31. Kaufman E, Lamster IB. The diagnostic applications of saliva-a review. Crit Rev Oral Biol Med. 2002;13:197-212.
32. Lamster IB, Kaufman E, Grbic JT, et al. ß-glucuronidase levels in saliva: Relationship to clinical periodontal parameters. J Periodontol. 2003;In Press.
33. Lamster IB, Pope MR. Reflections on the development of a host-response diagnostic test for periodontal disease. Technol Health Care. 1996;4:331-338.
34. Koizumi W, Tanabe S, Imaizumi H, et al. Effect of anti-Helicobacter pylori IgG antibody titer follwing eradication of Helicobacter pylori infection. Hepatogastroenterology. 2003;50:293-296.
35. Beck JD, Offenbacher S. The association between periodontal disease and cardiovascular diseases: A state-of-the-science review. Ann Periodontol. 2001;6:9-15.
Dr. Grbic received his DMD degree from the Fairleigh Dickinson University School of Dentistry. He received a Master of Medical Sciences Degree in oral biology and certificate in periodontology from the Harvard School of Dental Medicine and was a research fellow in the Laboratory of Surgical Immunology at Brigham and Women’s Hospital, Harvard Medical School. Since 1989, Dr. Grbic has been on the faculty of the Columbia University School of Dental and Oral Surgery, where he currently is an associate
professor of clinical dentistry and director of the division of oral biology and the Center for Clinical Research in Dentistry. He has published over 90 papers, abstracts, and book chapters in the areas of immunology, risk assessment, periodontal diagnostics, and oral
manifestations of HIV. His current interests include the relationship of periodontal and systemic diseases as well as the pharmacotherapuetic management of periodontal diseases. Dr. Grbic maintains a private practice limited to periodontics in New York City.
Dr. Engebretson earned the Doctor of Dental Medicine cum lauda et thesis propria from the Harvard School of Dental Medicine. He spent 1 year as a visiting scientist at the Bronx VA Medical Center followed by postgraduate training in periodontics at Columbia University earning the Master of Science degree. Dr. Engebretson joined the faculty at Columbia and now teaches didactic and clinical periodontics at the graduate and undergraduate levels, and conducts clinical trials in periodontics. Dr. Engebretson is board eligible in the specialty of periodontics, is an act
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