The basis of periodontal therapy emanates from the first step in the process, that being the examination. Without a proper examination, a proper diagnosis cannot be made and treatment cannot commence. Although we still rely on our periodontal probe to provide much of the information we need to formulate a proper diagnosis, newer techniques are being investigated which will allow us to be more forward-thinking than retrospective in our approach to diagnosis. The following section will review some of the classic papers on examination using the periodontal probe and evolve into a section on the use of other parameters such as crevicular fluid and blood testing results to assist with the diagnosis of periodontal disease.
Periodontal Probe
The periodontal probe is still consider the instrument of choice in assessing the health of the periodontal tissues at the time of the examination. The measurement is based on the distance from the gingival margin to the coronal aspect of the junctional epithelium and measures the history of attachment loss up to the point in time of the examination. However, there are many factors that will influence that particular measurement. These factors include:
1. Probing reproducibility
2. Probing force
3. Probe angulation
4. Status of gingival health
5. Site location
6. Local anatomy
7. Type of probe used
Listgarten (1993) suggested that the true anatomic measurement of the “pocket depth” can only be determined histologically. He suggested the use of the term “probing depth” as a more correct term since it measures the depth of the pocket plus the inconsistent amount of connective tissue penetration. In 1992, Listgarten defined the sulcus as the distance from the free gingival margin to the coronal end of the junctional epithelium. However, the ability of the probe to accurately measure this distance has been questioned.
Probing Reproducibility
Isidor et al (J Clin Perio 1984;11:663-668) looked at the “Reproducibility of pocket depth and attachment level measurements when using a flexible splint” . Pocket depth and attachment level measurements were carried out on 17 patients with advanced periodontitis- twice before periodontal surgery and twice 3 months after periodontal surgery (at 3 week intervals). A flexible splint and periodontal probe with a diameter of 0.5 mm and gradations of 3-2-2-2-2-2-2 mm was used to measure lateral incisors, canines, and premolars as experimental teeth. Measurements were recorded when the probe could not be advanced further apically into the pocket using gentle pressure and were rounded off to the nearest higher millimeter. All measurements performed by the same examiner.
The results of the study showed that complete agreement was found between the first and second measurements, before as well as after surgery in approximately 60% of sites. For 95% of the surfaces, the first and second measurements differed by 1 mm or less. These difference never exceeded 3 mm. There was no statistically significant difference in reproducibility before or after surgery and the location of the probing did not influence the measurements produced.
Probing force also has a significant influence of the reproducibility of pocket depth measurements. Van der Velden and de Vries, (J Clin Perio 1980;7:414-420) performed a study with 3 different examiners. They used a standardized pressure-controlled probe (0.75 N )and found that this did not lead to any more reproducible pocket depth measurements than a conventional periodontal probe. However, when using variable pressures in their probing (0.15N, 0.25N, 0.50N, and 0.75N) van der Velden and de Vries (J Clin Perio 1978;5:188-197) found there to be a positive relationship between probing force and probe depth (mean pocket depth increased from 2.08 @ .15N to 3.71 @ .75N). van der Velden also looked at condemned teeth with a pressure sensitive probe to determine the optimal tip diameter to assess probing depth (J Clin Perio 1979;6:106-114). He concluded that using a probe of 0.63 mm diameter is optimal when using a force of 0.75N.
Probing Force
A recent article by Garnick and Silverstein (J Periodontol. 2000;71(1):96-103) reviewed factors associated with probing force, suggesting that the pressure exerted by the probe is proportional to both the diameter of the probe tip (d) and the force (F) used: pressure = F/µ(d/2)2. If the Force of probing is doubled, the pressure increases by a factor of 2. However, if the diameter of the probe (d) is doubled, the pressure is reduced by a factor of 4. Probes most commonly used today were developed by Ramfjord in 1959 who designed a round probe with a tip diameter of 0.4mm, which was accepted as the standard . This group suggests that although there has been a greater effort to standardize force , in fact probe diameter should be standardized as it appears to have a greater clinical significance.
One of the methods believed to assist clinicians in standardizing their examinations is the use of a constant force probe. Many studies have investigated the accuracy and reproducibility of constant force probes versus manual probes. In one such study, Arujo et al (J. Periodontology 2003) performed a clinical trial testing the variability noted when using of a standard force probe between three examiners. Inter-examiner reliability is important for reproducibility purposes in research groups. We are not told the name of the probe, but the measure was set to record the PD to within 0.1mm at 20g (0.25N) of force. 20 subjects were examined by 3 examiners ( 1 dentist, 2 hygienists) . Only healthy subjects were examined. Exclusion criteria included current (<6months ago) periodontal therapy, PD>3mm, on antibiotics or requiring prophylactic antibiotics. After calibration with the use of the probe, patients were measured for pocket depths. Standard errors were calculated for each of the examiners (0.38mm, 0.39mm and 0.42mm) with an average of 0.4mm (+/- 0.02mm) at the subject level. Inter-examiner variability averaged to 0.16mm (+/- 0.02mm). The conclusion here is that all three examiners were able to obtain a reproducible result with the constant force probe. However, what was not taken into consideration was that the patients examined are otherwise healthy, such that different results can be obtained with inflamed tissues.
A study that looked at a direct comparison between and automated and manual probe in a “disease” population was the study done by Oringer et al. (J. Periodontol. 1997). It was a prospective study comparing the reliability and variation in a manual (MP) vs automated (AP) periodontal probe. 46 adults with moderate to severe periodontitis had 411 sites examined at baseline and after 6 months. Baseline measures with a manual probe were taken and over the course of a week, AP and MP probings were done. They found less variability over the course of a week for the MP. However, after 6 months, the AP had less variability compared to the MP. The clinical relevance of this work involves comparing measures in detecting disease progress. Issues with site inflammation and bleeding often arise when completing these types of studies and can vary with forces. In this case a single examiner was used. Possible sources of error include the shape of the AP and difficulty in positioning the tip at the desired site to the desired depth. The other is that greater force may be applied with a MP compared to an AP. The results of this study show that an AP may have less variability of attachment level measures over a 6 month period and may be influenced less by local inflammatory factors. Thus instruments with less measurement variability allows for earlier detection of disease progression.
Type of Probe Used
Vartoukian et al (J Clin. Periodontol. 2004) evaluate a new periodontal probe tip design This a clinical and in vitro study of a periodontal probe with a modified tip. Instead of a circular tip, the tip is wide and flat. The reason for this design is to reduce the penetration of the probe through the connective tissue attachment (CTA). One reason to consider changing the design is that a standard probe has a tendency to penetrate the CTA if the tissue is inflamed, giving an erroneous reading. The new probe has a 1mm wide and 0.45mm flattened tip, compared to a standard 0.5mm circular tip probe. The probe tips were attached to a pressure sensitive probing device in order to deliver 0.25M of force, but manual readings were taken. 27 subjects (smokers-11 and non-smkers-16, 12M:15F) scheduled to have teeth extracted were tested. The patients were blinded to the type of probe used. They were also asked about the level of discomfort with the probing (assessed via a visual analog scale (VAS)). Measures were taken to the nearest 0.5mm and the extracted teeth were measure to the nearest 0.1mm from a mark placed at 6 sites to the microscopically examined CTA. BOP as a dichotomous measure was taken at 30s after probing. In vitro findings showed that the test probe had a greater distribution of forces compared to the standard probe. The test probe was within 0.5mm of the post extraction findings 72% of the time compared to 54% with the standard probe. There was a 0.4mm reading difference between the two probes, with the standard probe reading deeper. The use of the control probe resulted in less BOP (37.6%) compared to the standard probe (46.4%). Additionally, the test probe had similar readings whether the tissue was or was not inflamed. The test probe was found coronal to the CTA while the standard probe was found 0.27mm apical to the CTA. It was determine that both probes are reliable and provide reproducible results. However, there appears to be values closer to the true CTA and greater patient comfort as per a VAS with the new probe.
BLEEDING ON PROBING
Despite its limitations, bleeding on probing remains an integral part of a routine periodontal examination. Studies (Lang et al, J Clin. Perio 1990) have demonstrated that, in fact, the lack of bleeding on probing is a better predictor of gingival health than the presence of bleeding on probing as a predictor of disease status. Several indicies over the years have been established to measure and assess the level of gingival inflammation. Below are some recent studies of this subject.
Lang, et al (Periodontology 2000, 1996) published a review that looked at the use of bleeding on probing as a monitoring tool during supportive periodontal therapy.
He suggested that there is no accepted level for prevalence of bleeding on probing (BOP) above which the risk of disease recurrence has been established. However, a BOP of 25% is the cutoff point between patients with maintained periodontal stability and those with recurrent disease after 4 years. Progression of periodontal disease is defined by a clinical attachment loss (CAL) of 2mm or more. Approximately 4% of all sites lose attachment of which half were due to periodontal disease. 2/3 of these sites had BOP >30%. One in five of the sites had <20% BOP. In individuals undergoing supportive periodontal therapy, BOP <10% may be considered low risk, whereas BOP >30% should be scheduled for greater supportive care. However, it must be remembered that BOP is has an almost linear relationship to the forces applied. If forces higher than 20g/25N are applied, bleeding can be induced due to trauma rather than inflammation.
In this prospective study on the progression of gingivitis, 65 s(31 completed the study) systemically healthy participants (military) aged 19-30 were evaluated at baseline and 6 months later on their probing depths, Clinical attachment levels, recession and bleeding on probing. Smoking status (non vs current) were contrasted during a 6 month period of no periodontal intervention. Patients were monitored every 2 months. Data of OH routine and smoking were collected via questionnaire.
From this table we can see that the smoking group had higher numbers in the %BOP, % plaque at baseline. These values more than double over the course of 6 months, with the smokers faring worse than the non-smokers. As to why they accumulated more plaque on their teeth was not evaluated. However, it appeasr that the level of BOP (via multivariate regression) is influeced by the smoking status more so than the plaque status.
PLAQUE/CALCULUS
It is evident that plaque and calculus play a major role in the pathogenesis of periodontal disease. In 1998, Albandar et al.(J Clin. Periodontol) looked at gingival inflammation and subgingival calculus as determinants in the progression of early-onset periodontal disease. 156 individuals who were 13-20 years old at baseline were examined 2 times during 6 years to assess the attachment loss, gingival state and the presence of dental calculus. Participants were grouped in to three categories, localized, generalized or incidental early-onset periodontitis. Of teeth with 0-2 mm attachment loss at the beginning of the study and which developed ≥ 3 mm attachment loss during the following 6 years, there were twice as many teeth with overt gingival inflammation, and 4 times more teeth with subgingival calculus at baseline than teeth without. Gingivitis and subgingival calculus when present at both examinations resulted in a stronger association with the development of new lesions than presence of these variables at baseline.
Teeth with gingivitis at baseline had a significantly higher mean attachment loss during 6 years than teeth without gingivitis and teeth with subgingival calculus at baseline had a significantly higher mean attachment loss than teeth without subgingival calculus. The presence of gingivitis and subgingival calculus at baseline and 6 years later was associated with the occurrence of even higher disease progression during this period. The association between gingival inflammation and subgingival calculus and the development and progression of attachment loss during the study period in the generalized and the localized EOP groups was significantly higher than the association in the incidental EOP group. However, in an appreciable percentage of the sites in all 3 groups, the presence of gingivitis and subgingival calculus at baseline and 6 years later was not associated with attachment-loss during 6 years. The results suggest a significant association between gingival inflammation and subgingival calculus and the development and progression of early onset periodontitis.
FUTURE EVALUATION METHODS
Our current methods of evaluation of periodontal disease aim at assessing a PAST HISTORY of disease. We extrapolate, based on this information, to attempt to alter the trajectory of future disease progression based on this assessment. However, recent research has attempted to elucidate current disease activity so that it can be treated and eradicated before it progresses to a measurable change in attachment level. The following section attempts to evaluate some of the literature investigating such tests as:
Subgingival temperature
Gingival Crevicular Fluid Contents
Neutrophil Activity
Salivary Enzyme Activity
MMP/Collagenase Levels
Peripheral C-Reactive Protein Levels
B-Glucuronidase Levels
Elastase Levels
Aspartate Aminotransferase Levels
Alkaline Phosphatase Levels
Gelatinase Levels
Genetic Testing
Prostaglandin E2 Levels
SUBGINGIVAL TEMPERATURE
It has been reported that elevated subgingival temperature is an indicator of periodontal disease. In 1997 Wolff et al (J. Clin. Periodontol) looked at subgingival temperature in relation to gingival crevicular fluid enzymes, cytokines, and subgingival plaque micro-organisms. The purpose of this cross-sectional study was to determine whether there was any relationship between subgingival temperature and GCF levels of neutrophil elastase (NE), myeloperoxidase (MPO), beta-glucuronidase (BG), interleukin -1 (IL-1), and interferon alpha (IFN) as well as to confirm an association of subgingival temperature with clinical parameters and specific subgingival plaque micro-organisms. 27 subjects with no recent periodontal therapy were selected for the study. Normal, gingivitis, and periodontitis sites were selected based on previous radiographic and clinical examination. The temperature was measured using PerioTemp®, which uses the difference between subgingival and sublingual temperature to assess the site. GCF (collected using paper filter) and subgingival plaque samples were also assessed. Subgingival plaque samples were assayed for Pi, Aa, Pg, Fusobacterium nucleatum, Eikenella corrodens. Measurements for GCF cytokines and enzymes done with ELISA. Clinical measures for probing depth, attachment loss, bleeding index, gingival index and plaque index were reflected for sites classified as healthy, gingivitis and periodontitis. Increasing subgingival temperature were detected with worsening of diagnosis: 34.53, 34.91, and 35.74C for healthy, gingivitis, and periodontitis sites respectively. A comparison between core temperature and subgingival temperature showed significant differences: 1.82C for control, 1.43C for gingivitis, and 0.6 for periodontitis. GCF volumes, enzyme and cytokine levels generally increased with worsening of diagnosis. There was a significant relationship between subgingival temperature and clinical parameters. There was also a modest relationship between subgingival temperature and GCF enzymes NE, MPO, BG. There was no relationship between subgingival temperature levels and GCF cytokines. There was also a positive correlation between subgingival temperature and E.corrodens and F nucleatum with little to no correlation with Pi, Pg, Aa. In general, this study suggests there is some correlation between subgingival temperature and factors associated with periodontal disease progression.
SALIVARY ENZYME ACTIVITY
Proteases in GCF promote periodontal breakdown by degrading collagen. Bader and Boyd (J. Clin. Periodontol. 1999) ) assessed the long-term monitoring of adult periodontitis patients in supportive periodontal therapy. Specifically, they attempted to correlate gingival crevicular fluid proteases with probing attachment loss. This was a longitudinal retrospective study to determine if a chair side assay for neutral protease activity in GCF could provide an early indication of site-specific disease activity. Patients from 1 periodontal practice, previously treated for periodontal disease, and who were in a maintenance program receiving 3-6 month recall visits were tested. Assay scores and clinical data were collected every 6 months for an average of 26 months. Data was collected from 71 sites in 38 patients which have a minimum 4mm of CAL. NPA assay (Periocheck by ACTech) measures the levels of ACTIVE neutral protease in GCF within 12 minutes. For the duration of the study, 50% of sites lost 1mm of attachment (active group) and 50% did not (inactive group). When the percentage of sites with BOP or positive NPA assay score are compared for the 2 groups over the course of the study, the active group showed a significantly higher proportion of positive test results than inactive sites. As a predictor of breakdown, the NPA assay had an accuracy of 94% and a risk ratio of 37.6 as compared to values of 58% and 1.5 for BOP. When only the subset of sites >7 mm were considered, the NPA assay had a calculated accuracy of 92% versus a value of 50% for BOP. Therefore it was concluded that the NPA assay appears to differentiate between bleeding at sites exhibiting only chronic inflammation with no attachment loss, and bleeding at sites undergoing active attachment loss.
GINGIVAL CREVICULAR FLUID CONTENT
Over the years, several elements within the crevicular fluid have been viewed as possible markers for either the presence/absence of disease activity or for assessing the progression or stability of the periodontal condition. For example, B-glucuronidase levels within the crevicular fluid are indicative of neutrophil influx into the area (Chung, J Clin. Perio. 1997, Albandar J Clin. Perio 1998). Lamster in 1997 (Annals of Periodontol.) evaluated an number of components of crevicular fluid and their utility as markers of periodontal disease status. Other factors such as cathepsin-G (Tervahartiala 1996), prostaglandins and leukotrienes, myeloperoxidase (Yamalik 2000), and calprotectin (Nakamura 2000) to name a few. Below are some articles that highlight the advances in this field.
b-GLUCURONIDASE LEVELS
Beta-glucuronidase (BG, a lysosomal acid hydrolase release from PMNs) in GCF provides an indication of neutrophil influx, and is significantly correlated with attachment loss. The purpose of the study done by Albandar (J Clin. Periodontol. 1998) was to test the hypotheses that 1) BG is elevated in patients with early-onset periodontitis (EOP), BG activity correlates with disease severity, and 2) BG levels may reflect the local bacterial challenge in the gingival crevice. Subjects were selected from the National Survey of Oral Health of United States Children (1986/87), which included children 13-17 years old. A clinical exam to measure attachment loss at mid and mesial buccal sites of all permanent teeth (except the wisdom teeth) was performed. Selection for EOP was based on the patient having at least 2 teeth with ≥3mm attachment loss, or at least one tooth with ≥4mm attachment loss. Control had no teeth having ≥3mm attachment loss and were matched to cases on gender, race, age, geographical location, urban/rural residence. 6 years later, subjects were invited back for a second exam, where attachment loss (AL) was measured at 6 sites/tooth, and subgingival plaque and GCF samples were taken. 249 subjects participated. These patients were classified into localized EOP (9.6%), generalized EOP (25.7%), incidental EOP (23.3%), and control (41.4%). At follow-up, these patients were between 19-25 years with a demographic breakdown of 72% African American, 12% Hispanic, 16% white. Subgingival plaque samples were analyzed for bacterial species Aa, Pi, Ec, Fn, C. rectus, T. denticola by DNA hybridization. GCF samples were analyzed for BG levels using a fluorometric assay. The 3 EOP groups had significantly higher frequency of sites showing BOP, deeper pockets, greater attachment loss with the generalized EOP showing the worst BOP, pockets and AL results. BG levels in the EOP groups were significantly higher than that found in the control groups. The highest BG level was seen in generalized EOP with the highest BG levels also significantly correlating with BOP (in localized and generalized EOP) and increasing probing depths. When looking at only 3mm pockets, BG levels were highest in generalized EOP patients and control had lowest. In the control population and incidental EOP, BG levels did not correlate with BOP. In general, BG level were higher in sites harboring high levels of each of the 7 microorganisms assessed. When the sites from all individuals were pooled, BG levels wer significantly higher in sites having high levels of Pg, Pi, and Td. Also in individuals with generalized/localized/incidental EOP, the BG level was significantly higher in sites with high numbers of these 3 bacteria than in sites with low numbers of bacteria.
In 1997, Chung, Grbic and Lamster (J. Clin. Periodontol.) studied the presence of Interleukin -8 (IL-8) and B-glucoronidase (BG) in gingival crevicular fluid and compared it to clinical parameters in the corresponding pockets in patients with advanced chronic periodontitis. 30 subjects with advanced chronic periodontitis were compared to 14 control patients. These patients had no subgingival treatment within the past 12 months and no periodontal surgery in the past 5 years. GCF was collected from all teeth except the wisdom teeth. Crevicular fluid was analyzed for the lysosomal enzyme BG (which is a marker for PMN influx into the gingival crevice) as well as Il-8 content through and ELISA assay. Clinical measures included probing depth, plaque, BOP. For the periodontitis patients, after collection of initial samples, they underwent SRP and OHI, after which GCF was collected again 2 weeks later. The results indicated that healthy subjects had significantly higher IL-8 concentration than periodontitis patients. The total BG in GCF was greater for the periodontitis patients than controls. After SRP in periodontitis patients, total BG decreased significantly from baseline, but IL-8 was inconsistent in its levels. A reduction in IL-8 appeared to be accompanied by a corresponding reduction in BG activity. As elevated BG activity in GCF has been associated with an increased risk for probing attachment loss, it may be considered as a potential marker for future attachment loss. However, there does not appear to be as strong a correlation with IL-8 levels.
In 2003, Lamster et al. (J. Periodontol.) looked at the use of b-glucuronidase in saliva as a possible marker of disease activity. 380 subjects, both healthy and those suffering from periodontitis were tested. Prior to a clinical exam, a sample of unstimulated whole saliva (for BG assay) and blood sample were obtained (for whole blood count). At least 50% of the subjects had at least one site that demonstrated greater than 5 mm attachment loss. A significant correlation was found between salivary BG and mean probing depth, mean gingival index, number of sites with PD>=5mm, and mean attachment loss. There was additional positive correlations with salivary BG, total WBC, number of neutrophils present. A logistic regression model showed that the odds ratio (OR) of having 1 site with >=5mm pocket is 3.96 if BG activity is >100 units (compared with 2.84 for former smokers and 3.26 for current smokers). As well, the odds ratio (OR) of having 4 sites with >=5mm pocket is 3.77 if BG activity is >100 units; (compared with 2.29 for former smokers and 3.15 for current smokers).From this, the results suggest that a significant association exists between periodontal clinical parameters and salivary BG activity, which can be a useful screening tool for periodontitis or for monitoring response to treatment.
NEUTROPHIL ASSAYS
Neutrophils are a critical component of the innate immune system and are required to provide early detection and phagocytosis of bacterial invaders. However, along with protecting the host, neutrophils also release liposomal enzymes and oxygen radicals that are destructive to the periodontal tissues. Using neutrophil activity as a measure of disease activity has been investigated for many years. : Sigusch et al. (J Clin. Perio 2001) looked to investigate and compare the chemotactic behavior of crevicular fluid PMN (CF-PMN) in three different forms of periodontitis including localized early onset periodontitis (LEOP), generalized early onset periodontitis (GEOP) and adult periodontitis (AP). Subgingival plaque samples (by using sterile paper points) and CF-PMN samples (by using crevicular washing technique) were obtained from two deepest sites of each quadrant in 7 LEOP subjects (mean age 19.6 y.o.), 11 GEOP subjects (mean age 33.4 y.o.) and 12 adult periodontitis (AP). Their microbial composition and CF-PMN chemotactic behavior were compared to their controls who were age and sex matched. The chemotactic activity in the adult periodontitis group was significantly higher compared to its control group (P<0.05). In contrast, LEOP and GEOP groups showed a significant reduction in chemotactic activity of CF-PMN compared to their control group (P<0.01). Therefore, the results from this study have replicated previous findings that LEOP and GEOP patients often exhibit deficiencies of PMN function. Furthermore, the reduced chemotactic response of CF-PMN observed in LEOP and GEOP subjects is probably combined with the strict association of these subjects with high numbers of Porphyromonas gingivalis and Capnocytophaga species demonstrated in their subgingival plaque samples.
Van Dyke’s group has published several articles on PMN activity in relation to periodontal disease activity and changes with treatment. One such study (Buchmann et al J Clin. Perio. 2002) aimed to stud, an array of four gingival crevicular fluid (GCF) components that characterize the GCF-associated inflammatory PMN responses in chronic periodontal disease. The hypothesis was examined that the local PMN responses in untreated and treated chronic periodontitis can be differentiated by GCF lysomal enzyme activities and elastase-alpha-1-proteinase inhibitor complex. A total of 9 subjects with chronic periodontitis (mean age of 40.2 y.o; 5M, 4F) were included in this study. Clinical parameters and markers of the PMN-derived inflammatory tissue response in GCF (harvested from 8 teeth in each patient (two per quadrant) at mesio or disto-interpproximal sites with probing depths between 6-8mm; paperpoints/periopaper stripTM ) were assessed before(a.k.a. baseline) and 6 months after surgical periodontal therapy (OHI, subgingival SRP under local anesthetic, Open flap debridement at sites with periodontal defect with probing depth>6mm). Myeloperoxidase (MPO), beta-N-acetyl-hexosaminidase (beta-NAH) and cathepsin D (CD) were analyzed as indicators of the PMN-associated host tissue destruction, and elastase-alpha-1proteinase inhibitor complex (alpha-1-EPI) as the major serum protein inactivating PMN elastase. Significant improvement in clinical parameters after the treatment was accompanied by significant reductions in activities of all lysosomal enzymes tested (i.e. MPO, beta NAH, CD) and significant reduction in activity of alpha-1-EPI (for details, please refer to the table .3&4 below). In conclusion, the clinical healing in chronic periodontal disease is associated with a down-regulation of the local PMN-response following periodontal therapy as characterized by a decrease in lysosomal enzyme activities and in the alpha-1-proteinase inhibitor complex in GCF.
Glogauer and Goldberg have looked at PMN phenotyping through variations in activity in a healthy, chronic periodontitis and a refractory periodontitis population (Johnstone et al., J Periodontol, 2007). Venous blood was obtained from 12 non-smoking patients who had been diagnosed with Refractory AP, 10 patients with chronic periodontitis who had responded to periodontal therapy (CP), and 13 periodontally healthy controls (HCs). Peripheral blood PMNs were loaded with dihydrorhodamine 123 and stimulated with phorbol 12-myristate 13-acetate (PMA) to measure the receptor-independent respiratory burst of these key immune cells. Phagocytosis via the complement and Fc-gamma receptors was also assessed. PMNs from patients with RAP displayed significantly increased PMA-induced oxygen radical production compared to those from the HC and CP patients. PMNs from RAP patients also displayed increased phagocytosis compared to those from the CP group. The findings demonstrated a larger receptor-independent respiratory burst and higher phagocytotic activity in PMNs derived from patients with RAP compared to PMNs derived from CP patients and periodontally HCs. It was speculated that the higher intrinsic intracellular activity of the nicotinamide adenine dinucleotide phosphate oxidase system may account for the continued periodontal breakdown, despite ongoing periodontal therapy in these challenging patients.
COLLAGENASE/MMP’S
Several studies have looked at correlating MMP activity with disease activity in treated and untreated situations, with mixed results. Bhide et al (J Periodontol 2000) looked at fluoregenic septapeptide matrix metalloproteinase assay to assess responses to periodontal treatment and found that this assay was only useful in cases of severe disease. As well, Dahan et al (J Clin Perio 2001) also found that when looking at MMP -1 and MMP -2 in both healthy and diseased gingival biopsy specimen, no correlations could be made.
However, Mancini et al (J Periodontol 1999), while working with McCulloch’s group showed that an assessment of MMP-8 and collagenase activity through a soluble biotinylated-collagen assay (SBA) was found to be a useful tool. This study included 125 adult periodontitis patients with 5 early onset periodontitis patients and one edentulous patient. Subjects were classified as having stable (history of previous treatment for periodontitis within the last 5 years but no detectable attachment loss of more than 2mm within the last 5 years), moderate (fewer than 10 pockets that were more than 5 mm in depth) and severe periodontitis (more than 10 pockets with more than 5mm and history of attachment loss in the previous year). Control patients (32) exhibited no sign of periodontitis good test of disease activity. Parotid saliva was collected directly from Stensen’s duct to assess the inhibition of collagenase activity by salivary components and was measured by SBA to determine if the inhibition of enzyme activity was due to exposure to saliva. Collagenase from peripheral blood neutrophils was used as a positive control for MMP-8 throughout the study. The study indicated that MMP-8 activity was 18 fold higher in severe periodontitis than in stable periodontitis. Therefore, as expected after periodontal treatment, collagenase activity was strongly reduced which corresponded to improvement in periodontal health and by a decrease in pockets of 5 mm or more depth. The suggested threshold is 80nano units collagenase activity in patients with severe periodontitis, which may be helpful for screening or diagnostic purposes. Active MMP-8 can be detected in GCF by the SBA and that the test is specific, simple, rapid and reproducible and could be used for treatment planning of patients with severe periodontitis.
A seemingly inventive idea was the use of chair side “test sticks” that could be used to monitor MMP-8 levels before and after treatment. Mantyla et al (J Perio Research 2003) tested 11 patients with adult periodontitis, 10 with gingivitis and 8 controls were involved in this study. Clinical periodontal examinations were performed in terms of PD, AL, BOP, GI and PI. The MMP-8 test stick is based on the immunochromatography principle that uses two monoclonal antibodies specific for different epitopes of MMP-8, which detects neutrophil and non-PMN type of MMP-8. The results suggested that the test sticks assessing GCF MMP-8 levels differentiated between periodontitis, gingivitis and healthy sites.
ELASTASE
Elastase is another enzyme important in the breakdown of elastin, which is another important component of the connective tissue associated with the periodontal ligament. In a 2 year cohort study, Eley and Cox (J. Clin. Periodontol., 1996) attempted to determine if there is a positive relation between gingival fluid (GF) elastase activity, concentration, and periodontal attachment/bone loss after 2 years. 75 patients with moderate periodontal disease were assessed. Significantly higher levels of enzyme activity were detected in sites that demonstrated rapid attachment loss over the two year study period. This study indicated a linear correlation between elastase activity and periodontal attachment loss.
These findings were confirmed in an animal study soon afterwards. Renvert et al (J. Clin. Periodontol. 1998) looked at a correlation between crevicular fluid elastase levels and histologically-observed attachment loss in beagle dogs. Three groups of dogs were observed, one that had their teeth brushed (healthy), one that was allowed natural plaque accumulation (gingivitis) and finally one with ligature-induced periodontitis. In the periodontitis model, the greatest rate of attachment loss (days 1-7) coincided with the highest levels of elastase activity. Thus this study also demonstrated a direct correlation between elastase levels and clinical attachment loss.
C-REACTIVE PROTEIN
C-Reactive protein is found in the blood and formed by the liver in response to inflammation. In 2001, Noack, Genco et al (J. Periodontol. 2001) looked at C-reactive protein levels in a well-characterized patient population. 174 patients were randomly selected and grouped based on their disease severity. Presence of P. ginigivalis, P. intermedia, C. rectus and B forsythus in subgingival plaque was measured by immunofluorescence microscopy. In reviewing the results, it was noted that there was an Increase in CRP levels in patients with periodontitis (4.1 mg/l) compared to healthy controls (1.7 mg/l). Participants with high levels of mean CAL had higher mean CRP levels than controls. As well, the presence of periodontal pathogens in subgingival samples was positively associated with elevated CRP levels. From this, the study showed that the extent of increase in CRP levels in periodontitis patients depends on the severity of the disease after adjusting for age, smoking, body mass index, triglycerides and cholesterol. There are elevated levels of CRP associated with infection with subgingival organisms often associated with periodontal disease.
In a 2006 study, Salzberg et al (J. Periodontol.) looked at C-reactive protein levels in an aggressive periodontal disease population. The group’s objective was to determine the relative levels of serum CRP in aggressive periodontitis (AgP). Serum samples were collected from three groups of patients, 93 patients with generalized AgP (GAgP), 97 patients with localized AgP (LAgP) and 91 healthy controls (non-periodontitis [NP]). Periodontal examination consisted of plaque index, gingival index, probing depth, bleeding index, and attachment loss measurements. Current smoking was assessed by determination of serum cotinine levels by enzyme-linked immunosorbent assay (ELISA), and serum CRP levels were determined using a high-sensitivity ELISA assay. The results indicated that the three groups were significantly different from one another in that the 95% confidence interval for serum CRP concentrations were as follows: NP, 0.6 (0.5 to 0.9); LAgP, 1.1 (0.8 to 1.5); and GAgP, 2.1 (1.5 to 2.7) mg/l. CRP levels in both LAgP and GAgP subjects were significantly greater than those in NP subjects, and levels in GAgP were significantly greater than those in LAgP. Following adjustment of the data for periodontal and demographic variables and current smoking, both mean probing depth and periodontal diagnosis remained correlated with CRP levels. Therefore, patients with AgP have statistically significant elevations in serum CRP levels compared to subjects without periodontitis. Elevated CRP in these subjects might represent a contribution of periodontal infections to systemic inflammation in relatively young individuals.
In order to assess whether or not the elevation in CRP was a result of the periodontal disease or was from other source, Yoshii, S., et al. (J Periodontol. 2009) looked for a temporal association of elevated C-reactive protein and periodontal disease in men. to ascertain the relationship between periodontal disease and CRP by following a large number of subjects for 1 year to clarify whether periodontal disease or elevated CRP was the precursor. 11162 men in Nagoya, Japan, age: 20-83 years participated in the study. The participants had an initial dental examination as part of a complete physical examination and then underwent the same examination 1 year later. Periodontal disease was defined as probing depth ≥ 4 mm. For the 4997 men without periodontal disease at baseline, logistic regression analysis was performed to examine the relationship between baseline CRP and periodontal disease 1 year later, adjusting for age, body mass index, glycosylated hemoglobin A1c level, and smoking status. Similarly, logistic regression analysis was performed to examine the relationship between periodontal disease at baseline and CRP 1 year later for the 10376 men with normal baseline CRP, adjusting for the same confounding factors. Among men without high CRP at baseline, periodontal disease at baseline correlated to CRP 1 year later. The odds ratio was 1.3 (95% confidence interval: 1.1 to 1.7). In the men without periodontal disease, no significant correlations were seen with baseline CRP or periodontal disease 1 year later. The odds ratio was 1.2 (95% CI: 0.9 to 1.5). Therefore, it was concluded that periodontal disease increased the risk for high serum CRP levels in men after 1 year of follow-up and that it was in fact the disease process that likely contributed to the change in CRP observed.
ASPARTATE AMINOTRANSFERASE
Although not commercially available at this time, the use of aspartate aminotransferase (AST) as a possible chairside diagnostic tool showed great promise in the late 1990’s. Oringer et al (J. Periodontol. 2003) published a well-designed paper on the relationship between crevicular aspartate aminotransferase levels and periodontal disease progression. This test aimed to use a chairside assay for monitoring AST levels in GCF and to examine the relationship between elevated AST levels and periodontal disease progression. Fifty-three subjects exhibiting moderate to severe chronic periodontitis (PS) and 15 periodontally healthy (HS) controls were recruited (mean age 48, male 39%). Over a 12-month period, 8 to 10 interproximal sites were monitored in PS and HS, monthly and quarterly, respectively for: GSF-AST (chairside test) and clinical parameters (BOP, PD, RAL- relative AL) At the 6- and 12-month visits, SRP was performed in the PS and prophylaxis in HS at 6 months. An automated probe used an occlusal disk as a landmark (mean of 2 measurements used or median of 3 if >0.5 mm between first 2 readings). Examiners were calibrated and inter-examiner reliability was assessed. AST semiquatitative test for 2 thresholds of positivity, 800 uIU and 1200 uIU, was performed. Disease progression was defined as an absolute change (1, 2 or 3 SD change from baseline vs 6 months. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were determined for each of 3 diagnostic criteria (AST > 800 uIU, AST > 1,200 uIU, and BOP) by using the 4 measures of disease progression. When a strict threshold for disease progression was utilized (RAL >1.74 mm), the AST >800 uIU threshold exhibited a 91.7% sensitivity. However, the specifcity was quite low at 34.5%. As expected, at the higher threshold of AST > 1,200 uIU, the sensitivity decreased to 58.3%, and the specifcity increased to 55.7%. Overall, the AST assay at both thresholds consistently demonstrated low PPV and high NPV regardless of the method used to define progression. In summary, there was a relatively weak association between a site exhibiting elevated baseline AST levels and subsequently experiencing attachment loss over the next 6 months. These findings suggest that the AST assay was associated with a large number of false-positive results. Positivity for AST does not provide the clinician with any significant diagnostic information in addition to BOP alone since many of the sites with a positive result do not exhibit disease progression.
RADIOGRAPHIC EVALUATION
Examination
A complete radiographic assessment is an important component of any routine periodontal evaluation. It is not until radiographs are combined with a full clinical assessment that an accurate diagnosis could be rendered. Pepelassi, and Diamanti-Kipioti (J. Clin. Periodontol 1997) attempted to determine the most accurate method of conventional radiography for the assessment of periodontal osseous destruction. Their goal was to compare surgical and radiographic measurements of osseous destruction as well as compare panoramic and periapical radiography in the assessment of periodontal osseous destruction. 100 patients with generalized moderate/severe periodontitis who would require periodontal surgery were recruited for the study. Baseline clinical examination was performed- PD and CAL at 6 sites per tooth (if detection of CEJ was not possible, used margin of restoration/prosthesis). Before treatment, each patient had a full-mouth series of radiographs and panoramic radiograph made. When flap surgery was performed, before any osseous resection was completed, linear measurements were taken to assess bone levels (“surgical measurements” represent gold standard, and only teeth treated surgically were included in study for a total of 2536 teeth/5072 proximal surfaces). Radiographs were assessed using the direct method (measuring from CEJ to the alveolar crest) and indirect methods (proportional % of osseous destruction and fraction of total root length). Osseous destruction was classified as being small (1-4 mm from CEJ), moderate (5-9 mm from CEJ), or severe (≥10 mm from CEJ). The surgical evaluation revealed that 1966 (38.76%) of proximal surfaces had small osseous destruction, 2744 (54.19%) had moderate destruction, and 359 (7.09%) had severe destruction. Periapical radiographs detected osseous destruction in 75 (3.81%) of the 1966 with small destruction while panoramic radiography only detected 16 (0.81%). PA’s were more accurate in osseous destruction detection than PAN’s regardless of location of the dental surface and the degree of destruction. Radiographs tended to underestimate destruction in mild destruction, were relatively accurate with moderate destruction, and tended to overestimate destruction in severe cases. PAs and PANs were most similar in assessing severe destruction and least similar in assessing initial destruction. Therefore, PAs were 4.7x times more successful than PANs in detection of small defects, and should therefore be preferably used for this purpose. The selection of methods is less important in severe sites, as both appear to similarly overestimate destruction.
Tugnait, et al. (J of Dentistry, 2006) wanted to validate a model to assess the role of radiographs in the diagnosis and treatment planning of periodontal diseases. The purpose of the study was to investigate the value added to the clinical examination by radiographs and to review this against a direct clinical exam with the patient present. Patients underwent a basic periodontal examination (BPE), and were prescribed radiographs taken after the clinical examination.
201 patients were randomly assigned to one of 4 groups (different clinical investigations):
Based on this assessment, diagnoses and treatment plan decisions called the “real patient clinical assessment” (real cl) were made. The radiographs were then revealed and this used together with the clinical exam to form the “real patient clinical plus radiographic assessment” (real cl + rd). The clinical data for every patient was transcribed to create the “paper patient” records for the indirect method of assessment- this constituted the data set for “clinical information only” (paper cl). The radiographs and radiographic report for each patient were also used to create the “radiographic information only” (paper rd). The results indicated that there was substantial agreement between periodontal diagnoses made from the real and paper clinical assessments (kappa = 0.68). Little or no difference was seen for treatment of toothbrushing instruction, OHI, denture hygiene, smoking cessation, treatment of dentinal sensitivity, SRP, removal of overhang restorations, provision of a restoration, observation of a tooth. In other words, the radiographs had little influence on this treatment decision. However, greater differences were seen in the planned treatments of extractions and proposed surgery- substantially more individual teeth were scheduled for extractions when real patient data were available (244) as opposed to only paper records (132); 11 subjects were identified as needing surgery by real cl and 5 were planned through paper cl. The strength of agreement of periodontal diagnoses varied from group 1 (k = 0.49), 2 (k=76), 3 (0.56), and 4 (0.82). Interestingly, the addition of radiographs (real cl +rd) had less of an impact when more clinical information was available (Group 1 k= 0.38; Group 2 k = 0.61; Group 3 = 0.78; Group 4 k=0.79)
For real patients many more decisions to extract teeth were taken by clinical assessment compared with paper patients, which suggests a loss of detail in the paper format which had an impact on the decision to extract (i.e. possibly patient attitude towards tooth retention). There was less difference between the periodontal diagnoses made with and without radiographs when a greater level of clinical information was available for paper assessments. Therefore, Group 2 assessment appeared to give sufficient clinical information for patient management and may be an appropriate choice for initial diagnosis and treatment planning of periodontal patients in a general practice setting.
