ADVANCED DIAGNOSTIC AIDS

CONTENTSINTRODUCTIONADVANCES IN CLINICAL DIAGNOSISADVANCES IN RADIOGRAPHIC MEASUREMENTADVANCES IN MICROBIOLOGIC ANALYSISADVANCES IN CHARACTERIZING HOST RESPONSEADVANCES IN GENETIC ASSESSMENTADVANCED DIAGNOSTIC AIDS IN DETECTING HALITOSISADVANCED DIAGNOSTIC TOOL TO STUDY OCCLUSAL STRESSESCONCLUSION

IntroductionPeriodontal diseases - prevalent human diseases defined by the signs and symptoms of gingival inflammation and/or periodontal tissue destruction. Clinical diagnosis of periodontitis - measuring the loss of connective tissue attachment to the root surface (clinical attachment loss) and loss of alveolar bone (radiographic bone loss) . Provides evidence of past periodontal destruction, its extent and severity. Does not provide any information on the cause of the condition, on the patient's susceptibility to disease, whether the disease is progressing, whether it is in remission or whether the response to therapy will be positive or negative.Disease process itself is considered to be site specific and has a multifactorial originConsideration should be given to including microbiologic, immunologic, systemic, genetic, and behavioral factors, in addition to the traditional clinical and radiographic parameters, when assessing patient status.

ADVANCES IN CLINICAL DIAGNOSISGingival bleeding Clinical evaluation - degree of gingival inflammation - redness and swelling of the gingiva + gingival bleeding. Gingival bleeding is a sensitive clinical indicator of early gingival inflammation. Clinical advantage of being more objective. Good indicator of the presence of an inflammatory lesion in the connective tissue

Severity of bleeding increases increase in size of the inflammatory infiltrate.

Evaluated- periodontal probe or a wooden interdental cleaner .

Gingival bleeding universally considered indicator of gingival inflammation and, by some indicator of disease activity.

Relationship to disease progression- unclear

Lang et al - retrospective study, reported that sites that bled on probing at several visits had a higher probability of losing attachment than those that bled at one visit or did not bleed. well controlled longitudinal studies investigated the predictive values of such clinical signs, trying to correlate them with attachment loss, but failed to demonstrate a significant correlation between bleeding on probing and other clinical signs and subsequent loss of attachment.Limitation - healthy sites may bleed on probing. any force greater than 0.25 N evoke bleeding

Gingival Temperature :Kung et al - thermal probes are sensitive diagnostic devices - early inflammatory changes in the gingival tissues. Studies - suspected active periodontitis lesions can create measurable elevations in sulcular temperature. PerioTemp probe (Abiodent, Inc., Danvers, Mass) - pocket temperature differences of 0.10 C.A naturally- temperature gradient - between maxillary and mandibular teeth and between posterior and anterior teeth.

Individual temperature differences are compared with those expected for each tooth.

Higher temperature pockets are signalled with a red emitting diode.

Haffajee et al used this probe to assess its predictability in identifying loss of attachment, concluding that sites with a red (higher) temperature indication had more than twice the risk for future attachment loss than did those with a green indication. However, the influence of pocket depth on temperature is still not clear, and further studies are needed to demonstrate the accuracy of this device and its utility in clinical diagnosis.

Periodontal Probing Classification a) first generation probes/ conventional b) second generation probes/pressure sensitive c) third generation probes/ computerized

Most widely used diagnostic tool - clinical assessment of connective tissue destruction in periodontitis . Increased probing depth and loss of clinical attachment are pathognomic for periodontitis.. Use of the periodontal probe - many problems in terms of sensitivity and reproducibility of the measurements. Clinical pocket depth obtained - not normally coincide with the histologic pocket depth. Tissue inflamed- offers less resistance to probe penetration, and the probe tip either coincides with or is apical to the coronal level of connective tissue attachment. Healed gingiva - subgingival instrumentation -increased resistance to periodontal probing.

Disparity - measurements depends on the probing technique, probing force, size of the probe, angle of insertion of the probe, and precision of the probe calibration. All of these variables contribute to the large standard deviations (0.5 to 1.3 mm) in clinical probing results, which make detection of small changes difficult.Mid 1980s, different probe prototypes -developed and tested to overcome these limitations. One of the main problems in reproducibility - variation in probing force. Penetration of the probe - positively correlated with probing force.

Solved - development of pressure sensitive probe. Studies have shown that with forces of up to 30 g, the tip of the probe seems to remain within the junctional epithelium, and forces of up to 50 g are necessary to diagnose periodontal osseous defects. Standardization of probe tips (less than 1 mm) and use of registration stents - reproducible probing angulation - used to overcome sources of error. Fabrication of stents - time consuming and impractical for clinical diagnosis.

Gibbs et al - developed Florida Probe System (Florida Probe Corporation) Probe tip - 0.4 mm in diameter. This probe tip reciprocates through a sleeve, and the edge of the sleeve provides a reference by which measurements are made. These measurements are made electronically and transferred automatically to the computer when the foot switch is pressed. Constant probing force is provided by coil spring inside the probe hand piece and digital readout. Advantages constant probing force precise electronic measurement computer storage of dataDis-adv Lack tactile sensitivity - independent movement, which forces the operator to predetermine an insertion point and angle. Use of a fixed force setting throughout the mouth, regardless of the site or inflammatory status, may generate inaccurate measurements or patient discomfort. One common problem reported in different studies where the Florida Probe System has been compared with conventional probing is the underestimation of deep probing depths by the automated probe.

In fact, several studies have suggested that the use of this automated probing system does no offer any advantage over conventional probing, rendering a similar level of reproducibility.

However, other studies have clearly shown that with the use of trained operators and performing the double pass method the measurements taken with the Florida Probe System are significantly less variable (lower standard deviation) than those obtained with a conventional probe.

Magnusson et al obtained mean standard deviations (reproducibility) for clinical attachment level measurements of about 0.3 mm, which is clearly superior to an average of 0.82 mm, with a range of 0.52 to 1.30 mm reported by Haffajee et al using manual probing.

An electronic probe using an optical encoder transduction element - evaluated by Goodson and Kondon.

These authors reported somewhat higher reproducibility with the electronic probe compared to conventional probing and a correlation coefficient of 0.82 between the two different methods.

This probe has now been named interprobe (Bausch and Lomb). They provide constant probing force, computer storage of data, and precise electronic management of the resulting inflammation.

Clinical evaluations of these systems- reported slightly improved probing, although not clinically significant

The electronic probe described - Jeffcoat et al (the Foster Miller probe)Capable of coupling pocket depth measurement with the detection of the cemento enamel junction from which the clinical attachment level is automatically detected. Never - released for general use.

Researchers at the University of Toronto have also described a probe (the Toronto Automated probe) that, like the Florida probe, uses the occlusal incisal surface to measure relative clinical attachment levels. The sulcus is probed with a 0.5 mm nickel titanium wire - extended under air pressure. Controls angular discrepancies - mercury tilt sensor that limits angulation within 30 degrees, but it requires reproducible positioning of the patients head and cannot easily measure second or third molars.

Two other electronic probes (the Birek Probe and the Florida Disk Probe) have been designed to measure changes in attachment level using the occlusal surface or the incisal edge as a reference point. The Birek Probe works on constant air pressure and measures attachment level from the occlusal surface. In a study of duplicate measurements in nine subjects, it was found that 82% of the measurements were within a 1 mm difference.

Low study compared- two models of the Florida Probe ; the stent model and the new disk model. The two probes are similar in design (both have a probe tip diameter of 0.4 mm and are preset at a constant 25-g force) but differ in the way they rest on a fixed reference point. The probe tip of the stent model runs through a metal sleeve which has a 1 mm collar that rests on the ledge of a fabricated vacuform stent. The disk model has a metal disk 11 mm in diameter that rests on the occulusal surface or incisal edge of the tooth. Both probes are connected to a digital readout. When the probe tip is in correct alignment with the tooth and the collar or disk is placed on the respective fixed reference point, a foot activated rheostat is depressed to enter the measurement (to 0.1 mm) automatically into a computer.

The new electronic probes appear to be superior to manual probes.

In the studies described the range of overall standard deviations for repeated measurements of individual sites in different subjects was 0.17 to 0.32 mm. Regarding the ability to detect significant attachment level changes, this should still be an improvement over the 0.82 mm obtained by Haffajee et al using the manual probe.

Probes and the examination of patients considered for implants Examination of patients - considered for implants includes both clinical evaluation of soft tissues and a radiographic evaluation.. Probing around implants is difficult (1) the prosthetic construction may need to be removed for access (2) standard metal instruments are unsuitable. Instead, plastic or titanium probe tips should be used to avoid damage of the implant / tissue interface. If automatic probing is considered, the Florida Probe is available with a titanium tip that will not hurt the implant ; also, the Interprobe system comes with disposable plastic tips.

ADVANCES IN RADIOGRAPHIC MEASUREMENT

Radiographs cannot accurately reflect - bone morphology buccally and lingually provide -useful information on interproximal bone levels.

Provide information on the periodontium that cannot be obtained by any other noninvasive methods (e.g., root length, root proximity and presence of periapical lesions and estimates of remaining alveolar bone). Substantial volumes of alveolar bone must be destroyed before the loss is detectable in radiographs, specifically, more than 30% of the bone mass at the alveolar crest must be lost for a change in bone height to be recognized on radiographs.

Therefore conventional radiographs are very specific, but lack sensitivity.

This low degree of sensitivity - due to the subjectivity of radiographic assessment and to the inherent sources of variability affecting the conventional radiographic technique- (1) variations in projection geometry ; (2) variations in contrast and density due to differences in film processing, voltage and exposure time ; and (3) masking of osseous changes by other anatomic structures.

The variations in projection geometry - reduced by the use of well standardized long cone parallel radiographic techniques.

To standardize the radiographic assessment, radiographs should be obtained in a constant and reproducible plane, using film holders with a template containing some kind of impression material, which is placed in a constant position on a group of teeth, and an extension arm that can be precisely attached to both the film holder and the x ray tube. The use of a parallel radiographic technique should be standard to all radiographic assessments for periodontal diagnosis. The use of individualized film holders has been shown to be valid in evaluating bone changes in longitudinal studies and clinical trials.

Digital radiography :Variations in image quality - variables inherent to conventional radiography - reduced with the use of digital intraoral radiography.

Digital radiography enables the use of computerized images - stored, manipulated, and corrected for under and overexposures.

Yield almost equal image properties compared with conventional radiographs, but through digital storage and processing, diagnostic information can be enhanced.

Dose reduction obtained with this technique (between 1/3 to of dose reduction compared with conventional radiographs).

Two digital radiography systems rely on the sensor - the direct and indirect methods The direct method uses a change coupled device (CCD) sensor linked with a fiber optic or other wire to the computer system.

This direct digital radiography - real time imaging, offering both the clinician and the patient an improved visualization of the periodontium by image manipulation and comparison with previously stored images.

Disadvantage - limited sensor area, which is only large enough to depict one or two teeth. The sensor rigidity attached to a wire- sterility issues- ideal image projection by using film holders very difficult.

The indirect method (Digora System) - phosphor luminescence plate, which is a flexible film like radiation energy sensor placed intraorally and exposed to conventional x ray tubes. A laser scanner reads the exposed plates offline and reveals digital image data, which can be enhanced, stored, and compared with previous images.

Advantage of this indirect method is due to the plate size and flexibility - almost identical to conventional x ray films - paralleling technique with the use of film holders can be easily applied.

Digital intraoral radiography is in a state of rapid development. Sensors, as well as computer hardware and software, are continually modified and improved.

Due to the clear advantage of real or almost real images that can be improved and to the important educational component of online images presented to the patient, it is expected that digital radiography will soon replace conventional radiography in modern daily practice.

However, certain improvements should be expected in order to overcome some of the current limitations.

Detecting Osseous Changes Between Radiographic Examinations :All radiographic methods simply image the existing anatomy of the teeth and supporting tissue- do not indicate the rate or presence of active bone or attachment loss, nor do they indicate whether past episodes of destruction and healing have occurred. To do so, the clinician must compare two or more carefully exposed radiographs taken at different examinations. Most assessment of bone loss in clinical practice today is achieved by visual comparison of the radiographs. The radiograph contains so much information that it is difficult for the human eye to detect small changes in bone support in the presence of a busy background containing the teeth and cortical and trabecular bone. Studies have shown that a 30% to 50% change in bone mineral is needed to be visible even to the experienced clinician using interpretative radiography.Several methods have been developed to allow measurement of bone height on serial radiographs. The simplest method involves measuring the distance from the CEJ to the alveolar crest. Of course, this method is limited both by the geometry of the film and the ability of the clinician to detect the alveolar crest. Methods have been developed that, in part, correct for errors in angulation. Bone loss may be expressed as a percentage of the root length to partially correct for errors due to foreshortening or elongation. This method, like the direct measurement method, is also limited by the ability of the clinical to accurately detect the alveolar crest. Nonetheless, such methods have substantial sensitivity in experienced hands. Changes in bone height as small as 0.1 mm may be detected.

Subtraction radiography :

A well established technique in medicine - introduced as a technique in periodontal diagnosis.

Shown (1) a high degree of correlation between changes in alveolar bone determined by subtraction radiography and attachment level changes in periodontal patients after therapy and (2) increased detectability of small osseous lesions compared with the conventional radiographs from which the subtraction images are produced.

Grondahl et al, using subtraction analysis - perfect accuracy at a lesion depth corresponding to 0.49 mm of compact bone - three times larger to be detectable with a conventional radiology technique.

Shown a degree of sensitivity similar to that for I125 absorptiometry - detect a change in bone mass of as little as 5%. Subtraction radiography has been applied to longitudinal clinical studies. Hausmann et al detected significant differences in crestal bone height of 0.87 mm, and Jeffcoat et al showed a strong relationship between probing attachment loss detected using sequential measurements made with an automated periodontal probe and bone loss detected with digital subtraction radiography.

Disadvantage : need to be close to identical projection alignment during the exposure of the sequential radiographs, which makes this method very impractical in a clinical setting.

Recently, new image subtraction methods (diagnostic subtraction radiography (DSR) have been introduced combining the use of a positioning device during film exposure with specialized software designed for digital image subtraction using conventional personal computers in dental offices. This image analysis software system applies an algorithm that corrects for the effects of angular alignment discrepancies and provides some degree of flexibility in the imaging procedure.

Recently, the use of the DSR technique has been compared with the conventional subtraction radiography technique yielding statistically significant gains in diagnostic accuracy over conventional radiographs and no differences against the classical subtraction radiography technique

Use of subtraction radiography or CADIA presupposes that the radiographs are taken with similar contrast, density, and angulation.

Color coding the subtraction images improves the ability of the novice clinician to detect bone loss or gain. In the color images, bone gain - green and bone loss -as shades of red.

Computed Tomography :Specialized radiographic technique that allows visualization of planes or slices of interest. Unlike conventional tomography, which blurs all structures not in the plane of interest, computed tomography actually removes the structures not in the plane of interest, resulting in an image with a clear visualization of the slice through the organ (s) under study.To accomplish this, multiple exposures are taken using known geometry around the area of interest. Specialized hardware, known as the CT scanner, is used to accomplish this. Software is then used to reconstruct each plane or slice of interest. Since CT equipment is expensive and the procedure carries a relatively high radiation burden for the patient - use should be reserved for questions that cannot be answered via clinical examination and / or conventional transmission radiography.

Magnetic resonance imaging

Fundamentally different from the other imaging techniques and does not use ionizing radiation.

Unlike conventional radiographic techniques, the hard tissues, bone, and teeth are not strongly imaged The strength - ability to image soft tissue.

To acquire an MRI image the patient is placed in a strong magnetic field. The protons of the hydrogen nuclei of the water within the tissues precess (rotate like a spinning top) about the direction of the magnetic field. Resonance frequency energy is applied and then removed. The response of the nuclei to the resonance frequency stimulation is observed in a receiver coil.

Based on these principles it is not surprising to find that alveolar bone is only weakly imaged, whereas soft tissue, such as salivary glands, dental pulp, and the disc of the temporomandibular joint, may be easily observed if the correct scanning parameters are used. .

Advantages - noninvasive and we are presently unaware of significant biological risks.

Dis-advantages: expensive, requires considerable scan time for high resolution images, and may be claustrophobic for the patient. MRI is prone to some of the same errors as CT, including motion and volume averaging artifacts.

MRI is a rapidly evolving field and its usefulness to dental applications is under investigation

Nuclear Medicine Bone Scans

Branch of radiology that uses radiolabeled pharmaceuticals that are specifically intended to image particular organs or detect specific disease processes. For the diagnosis of periodontal disease, bone scans have been used to detect sites of active bone loss. The radiopharmaceutical is a technetium labeled disphosphonate called 99m Tc methylene diphosphonate.

The diphosphonate moiety is the bone seeker that is adsorbed onto the forming front of bone that occurs either during bone apposition or behind the bone resorption.

The radiolabel, technetium, is a synthetic element with a 6 hour physical half life. The biological half life of the radiopharmaceutical is 3 hours.

The bone scan does not image the anatomy of the area of interest. Rather, it detects alteration in bony metabolism that may occur prior to radiographically detectable changes.

It is also important to note that these changes are not specific to a particular disease. therefore, the bone scan must be used in conjunction with clinical findings and radiographs in order to make an accurate diagnosis.

To perform a bone scan, the radiopharmaceutical is injected intravenously. Following a period to allow for bony uptake of the agent, uptake is either imaged using a gamma camera or measured using specially designed detectors for intraoral use. Areas of active bone loss appear as hot spots in the image.

Studies have shown that the sensitivity and specificity of this technique in prognosticating alveolar bone loss due to periodontitis subsequently determined radiographically is over 90%.

Bone scans are best used to determine whether a patient has active sites of bone loss and could benefit from an experimental treatment, or to determine whether a patient who is to undergo a bone marrow transplant has sites of active periodontal disease or occult disease that need immediate attention.

Advances in Microbiologic Analysis :

Subgingival oral bacteria - main initiating agents in the development of periodontal disease.

These microbiologic tests - potential to support the diagnosis of the various forms of periodontal disease, to serve as indicators of disease initiation and progression (i.e., disease activity), and to determine which periodontal sites are at higher risk for active destruction.

Microbial tests can also be used to monitor periodontal therapy directed at the suppression or eradication of periodontopathic microorganisms.

Several methods have been employed for the detection of putative periodontal pathogens in subgingival samples. Some of these methods have been strictly used for research purposes, whereas others have been adapted or modified for clinical use.

All of these methods share the common need for an appropriate subgingival plaque sample.

Selecting the proper specimen site and collecting an adequate sample are essential elements in periodontal microbiology.

These samples may be difficult to obtain in patients infected by organisms that are unevenly distributed in the dentition.

Bacterial Culturing : Considered the reference method (gold standard) when determining the performance of new microbial diagnostic methods. Plaque samples are cultivated anaerobically and by using selective and nonselective media, together with several biochemical and physical tests, the different putative pathogens can be identified.

Advantage :Obtain relative and absolute counts of the cultured species.

Only in vitro method - assess for antibiotic susceptibility of the microbes

Shortcomings: Only grow live bacteria, therefore strict sampling and transport conditions are essential.

Putative pathogens, such as Treponemas sp. and Bacterioides forsythus are fastidious and difficult to culture.

Sensitivity of culture methods is rather low, since the detection limits for selective and nonselective media average 103 to 104 bacteria and hence low numbers of a specific pathogen in a pocket - undetected.

Important drawback is that culture requires sophisticated equipment and experienced personnel and is relatively time consuming and expensive.

Clinicians, when using this methods, must be confident that the laboratory has the appropriate technology and expertise in periodontal microbiology to communicate diagnostically and therapeutically useful information to them.

Direct microscopy

Darkfield or phase contrast microscopy - suggested as an alternative to culture methods - ability to directly and rapidly assess the morphology and motility of bacteria in plaque sample.

Used to indicate periodontal disease status and to structure maintenance programs.

However, most of the main putative periodontopathogens, including Actinobacillus actinomycetemcomitans, P. gingivalis, B, forsythus, Eikenella corrodens, and Eubacterium species, are non motile, and therefore this technique is unable to identify these species.

It is also unable to differentiate among the various species of Treponema.

Seems an unlikely candidate as a diagnostic test of destructive periodontal diseases

Immunodiagnostic Methods Immunological assays employ antibodies that recognize specific bacterial antigens to detect target microorganisms. Revealed using a variety of procedures: Direct and indirect immunofluorescent microscopy assays (IFA)Flow cytometryEnzyme linked immunoabsorbent assay (ELISA) Membrane assay Latex agglutination.

Direct IFA - monoclonal and polyclonal antibodies conjugated to a fluorescein marker that binds with the bacterial antigen to form a fluorescent immune complex detectable under a microscope. Indirect IFA employs a secondary fluorescein conjugated antibody that reacts with the primary antigen antibody complex. Both direct and indirect immunofluorescence assays are able to identify the pathogen and quantify the percentage of the pathogen directly using a plaque smear. IFA has been used mainly to detect A. actinomycetemocomitans and P. gingivalis.

Zambon et al - technique is comparable to bacterial culture in its ability to identify these pathogens in subgingival dental plaque samples. In fact immune fluorescence microscopy may be even more likely to detect them in clinical samples because it does not require viable bacterial cells.Comparative studies indicate that the sensitivity of these assays ranges from 82% to 100% for detection of A. actinomycetemocomitans and from 91% to 100% for detection of P. gingivalis, with specificity values of 88% to 92% and 87% to 89%, respectively.

Cytofluorography or flow cytometry - rapid identification of oral bacteria involves labeling bacterial cells from a patient plaque sample with both species specific antibody and a second fluorescein conjugated antibody. The suspension is then introduced into the flow cytometer, which separates the bacterial cells into an almost single cell suspension by means of a laminar flow through a narrow tube. The sophistication and cost involved in this procedure precludes its wide usage.

Similar in principle to other radioimmunoassays, but an enzymatically derived color reaction is substituted as the label in place of the radioisotope. The intensity of the color depends on the concentration of the antigen and is usually read photometrically for optimal quantitation . Used primarily to detect serum antibodies to periodontopathogens ; however it has also been used in research studies to quantify specific pathogens in subgingival samples using specific monoclonal anibodies. ELISA

Membrane immunoassay

Recently marketed (Evalusite).

It involves linkage between the antigen and a membrane bound antibody to form an immunocomplex that is later revealed through a colorimetric reaction.

Evalusite - designed to detect A. actinomycetemcomitans, P. gingivalis, and P. intermedia.

Immunological assays for oral bacteria, although extensively used for research purposes, lack the clinical validation since most of them have never been available commercially.

Cross reactivity leading to detection of false positives may represent a major problem, mostly when polyclonal antibodies are used. On the other hand the use of monoclonal antibodies may cuase the occurrence of false negatives when compared with culture, due to their high specificity.

Immunological assays can identify dead target cells, thus not requiring stringent sampling and transport methodology.

Cannot be used to determine antibiotic susceptibility.

Most of these assays provide a quantitative or semiquantitative estimate of target microorganisms ; however, these methods generally show poorer detection limits than nucleic acid probes of PCR assays.

Snyder et al tested the sensitivity of the Evalusite test, demonstrating a detection limit of 105 for A. actinomycetemcomitans and 106 for P. gingivalis.

Latex agglutination

Very simple immunological assay based on the binding of protein to latex. Latex beads are coated with the species specific antibody, and when these beads come in contact with the microbial cell surface antigens or antigen extracts, cross linking occurs ; its agglutination or clumping is then visible usually in 2 to 5 minutes. Because of their simplicity and rapidity, these assays have great potential for chariside detection of periodontal pathogens. Two types of latex agglutination tests the indirect assay and inhibition assay. The indirect assay is the most common latex agglutination test for bacteria.The antibody is bound to latex. When a suspension of the plaque sample is mixed with the sensitized latex and gently agitated for 3 to 5 minutes, resulting agglutination or clumping is indicative of a positive result for the bacteria being tested. The inhibition assay is based on the principle of inhibiting the expected agglutination reaction between known antigen and known antibody as a result of competition.

Enzymatic Methods of Bacterial Identification B. forsythus, P. gingivalis, the small spirochete Treponema denticola, and Capnocytophaga species share a common enzymatic profile, since all have in common a trypsin-like enzyme. The activity of this enzyme can be measured with the hydrolysis of the colorless substrate N benzoyl arginine 2 naphthylamide (BANA). When the hydrolysis takes place, it releases the chromophore - naphthylamide, which turns orange red when a drop of fast garnet is added to the solution. Diagnostic kits has been developed using this reaction for the identification of this bacteria profile in plaque isolates (Perioscan). Loesche et al proposed the use of this BANA reaction in subgingival plaque samples to detect the presence of any of these periodontal pathogens and thus serve as a marker of disease activity. Using probing depths as a measure of periodontal morbidity, Loesche et al showed that shallow pockets exhibited only 10% positive BANA reactions, whereas deep pockets (7 mm) exhibited 80% to 90% positive BANA reactions.

Beck et al used the BANA test as a risk indicator for periodontal attachment loss. Taken collectively, results using this diagnostic method suggest that positive BANA findings are a good indication that T. denticola, P. gingivalis, or both are present at sampled sites. One of the potential difficulties of this test is that it may be positive at clinically healthy sites and remains to be proven whether this test can detect sites undergoing periodontal destruction. Besides, since it only detects a very limited number of pathogens, its negative result does not rule out the presence of other important periodontal pathogens.

BACTERIAL IDENTIFICATION BY TOXINS & PROTEINSTOPAS I Kit: The TOPAS I (Toxicity prescreening assay) is a chairside ; calorimetric assay designed to detect 2 markers of bacterial infection in gingival crevicular fluid i.e. toxins and proteins.Rationale of TOPAS I The approach used in TOPAS I is to react the Toxin and protein samples with a mixture of reagents to produce a new set of compounds which have one or more chromophores. In the case of toxins these chromophores all absorb visible light in the yellow (420 nm) region;whereas in the case of proteins all these chromophores absorb visible light in the blue (600 nm) region.

Toxin detection The toxins are produced by pathogenic anaerobic bacteria isolated from active periodontal disease sites which are measured at high (millimolar) levels in the GCF. TOPAS is calibrated using hydrogen sulfide as the toxic standard at levels ranging from 0 mM (noassay) to 2mM (extreme toxicity). The toxins react with a mixture of chemical reagents to produce yellow coloured by-products.Toxins + Colourless reagent -> Yellow Colour (bacterial)

Protein detection Proteins include both bacterial proteins as well as human inflammatorv and serum proteins such as antibodies and globulins. Bacterial infection increases the level of these proteins in the GCF.TOPAS is calibrated using Human serum albumin (HAS) as the protein standard at levels ranging from none detectable (50 ug ). The proteins react with the chemical reagents to produce blue colored by-products.

Proteins + Brown Dye -> Blue colour(antibodies + Albumins + Enzymes)

The colour intensity of the toxins and protein solution is graded by result chart.

Deoxyribonucleic Acid Probe Technology

Nucleic Acid Probes. Entail segments of single stranded nucleic acid, labeled with an enzyme or radioisotope, that can locate and bind to their complementary nucleic acid sequences with low cross reactivity to nontarget organisms.

Target whole genomic DNA or individual genes.

Whole genomic probes are more likely to cross react with nontarget microorganisms due to the presence of homologous sequences between different bacterial species.

However, specific genes, such as 16S rRNA (ribonucleic acid) genes, contain signature sequences limited to organisms of the same species.

These oligonucleotide probes display limited or no cross reactivity with nontarget microorganisms.

The assay can rapidly test for multiple bacteria, including A.actinomycetemcomitans, P. gingivalis, B. intermedius, C. rectus, E. corrodens, Fusobacterium nucleatum, and T. denticola in multiple clinical plaque samples. The probes are able to detect as few as 102 to 104 bacteria, and the sensitivity and specificity are not affected by the presence of unrelated bacterial in mixed culture samples.

Restriction Endonuclease Analysis Recognize and cleave double stranded DNA at specific base pair sequences. The DNA fragments generated are separated by electophoresis, stained with ethidium bromide, and visualized with ultraviolet light. The genetic heterogeneity and homogeneity of stains can then be evaluated by comparing the number and size (electrophoretic pattern) of the DNA fragments obtained. These DNA fragment patterns constitute a specific fingerprint to characterize each strain. Powerful tool for determining the distribution of a specific pathogenic strain throughout a population. This technology has also been applied to the molecular genetic analysis of the natural diversity of such oral bacteria as A. actinomycetemcomitans, P. gingivalis, P. intermedia, E. corrodens, F. nucleatum, and T. denticola and has been very useful in studying the transmission patterns of putative periodontal pathogens among family members.

Polymerase Chain Reaction Amplification of a region of DNA flanked by a selected primer specific for target species. The presence of specific amplification product indicates the presence of the target microorganisms. Among the different nucleic acid assays. Demonstrates the best detection limits, as few as five to ten cells and shows no cross reactivity under optimized amplification conditions. Different bacterial species may be detected simultaneously by multiplex PCR in which several distinct primer pairs, each specific for a given target microorganism are employed in a single tube amplification process.Drawback - relatively small aliquots are used for the amplification process. If this small quantity of the plaque sample does not contain the targeted microorganism, the assay will not detect it. Subgingival plaque may contain enzymes that can alter the amplification process.

Until recently, PCR assays were able just to assess the presence of the target microorganism.

Fujise et al have developed a quantitative PCR method.

PCR assays have the potential for being an ideal detection method of periodontal microorganisms.

It is relatively easy to perform and demonstrates excellent detection limits and little cross reactivity under optimal conditions.

Advances In Characterizing The Host Response : Diagnostic tests have been developed - measures of the inflammatory process to conventional clinical measures.

Provide information on the destructive process itself, current activity of the disease, rate of disease progression, patterns of destruction, extent and severity of future breakdown, and likely response to therapy.

Clinician would be able to better individualize his therapeutic approach, thus customizing the recommended treatment.

Assessment of the host response refers to the study of mediators, by immunologic or biochemical methods, that are recognized as part of the individuals response to the periodontal infection. These mediators are either specifically identified with the infection, such as antibody to a putative pathogen, or represent a less specific reaction like the local release of inflammatory mediators, host derived enzymes, or tissue breakdown products.

Source of Samples:

GCF - more than 40 components of GCF - studied. Divided into three main groups : host derived enzymes, tissue breakdown products, and inflammatory mediators.

Gingival crevicular cells

Saliva - easily collected and may contain both locally and systemically derived markers of periodontal disease.No saliva based diagnostic tests are available to be used in clinical practice. Proposed diagnostic markers in saliva include proteins and enzymes of host origin, phenotypic markers, host cells, hormones (cortisol), bacteria and bacterial products, volatile compounds and ions.

Blood serum

Blood cells

Urine

Immune response :

Antibody : total immunoglobulin and IgG subgroups -specific antibody or total Ig in GCF appears to be of no use in distinguishing between stable and progressive sites. Some evidence suggests that a reduction in specific antibody in serum and consequently GCF in patients with existing disease can place them at risk for further disease progression Complement

Inflammatory response

Arachidonic acid derivatives, eg prostaglandin E2 (PGE2)

Cytokines, eg IL1, IL-2, IL-4, IL-6, TNF-.

IL 1 and are present in inflamed gingiva. They are also present in GCF from patients with periodontitis and extremely low concentrations are found at healthy sites. Their levels were reduced following scaling and root planing but were not found to correlate with probing depth measurements.

TNF - is also present in GCF but does not correlate with probing depth or gingival inflammation and its total amount was inversely related to tissue inflammation

No significant differences in the mean level of IL 1 in refractory or stable patients but refractory sites in this patients group produced significantly more IL-6.

PGE2 levesl are low - health gingivitis - modest rise in GCF PGE2 - 32 and 53 ng/ ml Untreated periodontitis patients - significantly higher levels than gingivitis patients. Levels greater than 66 ng/ml were found to be predictive of further possible loss of attachment and this level was used as a cut of value in a positive and negative screening test.

Diagnostic tests :Although GCF PGE2 has considerable potential as a screening test for periodontal activity strangely no commercial efforts are currently underway to develop one. In this regard, it is now possible to assay GCF PGE2 with an ELISA assay using a monoclonal rabbit anit -PGE2 antibody.

Cytokines are also assayed using ELISA techniques which could be developed into chairside kits. However, at present the predictive ability of these markers is still in doubt. Thus, the most likely diagnostic marker of the inflammatory and immune factors described above is GCF PGE2.

Proteolytic enzymes CollagenaseElastaseCathepsin GCathepsin BCathepsin DDipeptidylpeptidases Tryptase Hydrolytic enzymes Aryl sulphatase - glucuronidaseAlkaline phosphataseAcid phosphataseMyeloperosidaseLysozyme Lactoferrin.

Host Derived Enzymes

COMMERCIAL DIAGNOSTIC KITS BASED ON GCF PROTEOLYTIC AND HYDROLYTIC ENZYME LEVELS Periocheck (ACTech) :

Detects the presence of neutral proteinases such as collagenase in GCF.

A paper strip is used to obtain a GCF sample. This strip is then placed in contact with a collagen gel to which a blue dye has been covalently bonded. This is then incubated at 430 C. If the neutral proteinases are present in the sample they will attack the collagen gel and release the blue dye. The released blue dye produces a blue colour in the strip, the intensity of which is proportional to the amount of enzyme present in the sample. The intensity and the area of the colour is then scored on a scale of 0 to 2 by comparing it with three standards on a colour card which is provided with the test kit.

Prognostik (Dentsply) This system detects the presence of the serine proteinase, elastase, in GCF samples. A GCF sample is collected on special paper strips which have been impregnated with the appropriate peptidyl derivative of 7 amino- trifluoromethyl coum arin (AFC). The substrate used is MeOSuc Ala-Ala- ProVal AFC which detects elastase and is linked to a fluorescent leaving group, AFC. If elastase is present the sample reacts with the substrate in 4 8 minutes releasing the fluorescent leaving group, AFC. This produces green fluorescence in the strip which can be seen under ultraviolet (UV) light using a UV light box. The intensity of the fluorescence is proportional to the amount of GCF in the sample and this is scored by comparing it with AFC standards

Pocket WatchCSA AST PP inorganic sulfiteSulfite ion reacts MGC- allowing pink colored rhabdomyine dye to see through.

Advantages :

Some eg cathepsin B, elastase, dipeptidyl peptidases II and IV, and - glucuronidase, are predictive of disease activity in longitudinal studies.Simple to use, particularly the colour detection systems.Can be read after a short time.Can be shown to the patient and related to the tooth site. Disadvantages :

The choice of the most appropriate biomarker may still be difficult at the present state of knowledge.There is difficulty in determining the sites to sample and when to sample them. If a moiety is associated with inflammation this may mask its association with destructive disease.No account of biological control mechanisms is taken in present tests. cost.

Periogard AST in GCF :Test kit using paper point GCF samples and calorimetric detection. The test kit consists of a tray with two test cells for each tooth and appropriate reagents for conducting the test. The strip containing the GCF sample is placed into suitable test well and two drops of one agent (10 mM Tris HCL with 0.067% fiction X 100, pH 6.0) is added. At the same time positive and negative control cells are prepared using strips provided. Two drops of a solution provided (260 mMl - aspartic acid ; 33 mM 2 oxogluteric acid ; 4.3 mM disodium EDTA, 1.6% polyvinylpyrolidone, 0.067% Triton X- 100, 2.7 mm sorbic acid in 100 MM Tris HCL, pH 6.0) are added to the wells and allowed to incubate at room temperature. After 9 minutes incubation the substrate / detection solution (1 mg fast red RC diazotised salt in 1% methanol, 0.06% Triton X 100, in 230 Mm Tris HCL, Ph 8.0) is mixed and two drops are added at 10 minutes. Five minutes later the test results can be read by eye by comparing the test well colour to the colour of the positve control. A colour of greater intensity to that of the negative control is scored as positive and one of lesser or equal intensity as a negative result. The test is designed to be positive at 800 mIU AST activity and negative at values , 800 mIU

Markers of connective tissue degradation :Soft tissue degradation products :Fibronectin Hydroxyproline containing peptidesGlycosaminoglycans Bone specific proteins:Osteonectin and bone phosphoprotein(N-Propeptide)OsteocalcinCross linked carboxyterminal telopeptide of type-I collagen

Methods of isolation and detection The detection of osteonectin requires the use of nitrocellulose strips as it cannot be recovered from conventional strips. In contrast, GCF for the detection of osteocalcin and ICTP was collected on conventional paper strips left in situ for 30 seconds. However, in some studies of osteocalcin and ICTP multiple strip collection was used employing two in succession for one minute at the same site for very short collection periods. Clearly, some standardistion of collection technique is required if the data from studies is to be compared.The biochemical techniques used to isolate and detect components in this section may be difficult to modify for chair side use. However monoclonal and polyclonal antibodies have been produced for osteonectin, ICTP and N- Propeptide. Using these, osteocalcin has been assayed with either ELISA or radio- immunoassays, osteonectin and N-propeptide with ELISA and ICTP with radio-imunoassays.

ADVANCES IN GENETIC ASSESSMENTThe periodontitis susceptibility trait test is the only genetic susceptibility test for severe periodontitis that is commercially available. It evaluates the simultaneous occurrence of allele 2 at the IL-1A +4845 and 1B +3954 loci. A patient with allele 2 at both loci is considered genotype positive and therefore more susceptible to develop severe periodontitis.Used to identify a predisposition to disease, treatment approaches and outcomes will still be influenced by environmental and behavioral factors. Gives no information on disease activity or susceptibility

ADVANCED DIAGNOSTIC AIDS IN DETECTING HALITOSIS

GAS CHROMATOGRAPHY

The connection between bad breath (halitosis) and certain volatile sulfur compounds (VSC) was first established by Tonzetich . He determined that certain anaerobes were the source of these compounds, primarily methyl mercaptan, hydrogen sulfide, and to a lesser extent, dimethyl sulfide.Oral chroma individually measures three volatile sulfur compounds :hydrogen sulfide, methyl mercaptan and dimethylsulfide and correlates each with cause. Ideal as a pre-examination, preventive dentistry device.

Graphic displays are ideal for communicating with patients regarding their oral health condition. Monitor patint improvement with historical displays. The OralChroma including analysis software, computer connection cable, an ample supply of plastic syringes for collecting breath samples

PROCEEDURE

HALIMETER

Gives a digital readout in parts per billion (ppb) VSC, which is not only quantitative, but is more accurate than the very subjective organoleptic method. Using a recorder, in conjunction with the Halimeter, offers a hard copy record for both the dentist and patient..The only function of the Halimeter is to serve as a reliable monitor for the measurement of VSC concentrations. If other chemical agents cause bad breath, and might exist in the breath sample, they may or may not respond in the Halimeter.

The Diamond Probe/Perio 2000 System is a dental device designed to detect sulfide concentrations of various forms in gingival sulci. The system combines a conventional Michigan O style dental probe with a sulfide sensor, which measures PD, BOP, and SUL, simultaneously

The micro-sulfide sensor responds to sulfide ions and measures metabolic-products of mainly anaerobic bacteria and, indirectly, bacterial activity.

Intended to be utilized as part of a total program encompassing a thorough history and physical examination of the patient.

Along with an organoleptic assessment, the quantitative nature of Halimeter data can serve as an excellent tool for following the progress of treating halitosis, and for archiving hard copy records, halimeter data by itself cannot affirm whether a breath problem exists - VSC levels can change with the time of day.

The dental practitioner is required to include the assessment of other diagnostic procedures prior to making a positive conclusion.

ADVANCED DIAGNOSTIC TOOL TO STUDY OCCLUSAL STRESSES

The finite element method is numerical method, which can be implemented to solve many problems.

First developed in 1956 for the analysis of aircraft structural problems.

After within a decade, the potential of the method for the solution of different types of applied sciences and engineering problems were recognized.

Over the years, the finite technique has been so well established that today it is considered to be one of the best methods for solving a wide variety of practical problems efficiently.

Application of fem

Finite element analysis as well as other related morphometric techniques such as the macroelement and the boundary integral equation method (BIE) is useful for the assessment of complex shape changes.

To the description of form changes in biological structures (morphometrics), particularly in the area of growth and development. For the understanding of stress related bone remodeling and also provides a guideline reference for the design of dental implants.

Useful for structures with inherent material homogeneity and potentially complicated shapes such as dental implants

Analysis of stresses produced in the periodontal ligament when subjected to orthodontic forces

To optimize the design of dental restorations

Advantages

It is a non-invasive method

The actual stress at any point can be measured.

The actual displacement (initial tooth mobility) of the tooth can be measured.

The reproducibility does not effect the physical properties of the involved material and the study can be repeated as many times as the operator wants.

CONCLUSION Although there are many potential markers for periodontal disease activity and progression, still neumerous features hamper the ability to use them as diagnostic tests of proven utility. There is still a lack of a proven gold standard of disease progression and thus the correlation of these potential markers with proven clinical attachment loss may be a potential confounder in any proposed test. After all these years of intensive research we still lack a proven diagnostic test that has demonstrated high predictive value for disease progression, has a proven impact on disease incidence and prevalence and is simple, safe and cost effective.

REFERENCES:

Clinical Periodontology by Newman 8th & 9th Edition.Diagnosis & Risk Prediction of Periodontal DiseasesVol 3- Per Axelsson.Periodontics Medicine, Surgery and Implants RoseOutline of Periodontics - 4th edition JD Manson & EleyAdvances in Periodontics Wilson, Kornmn & NewmannFundamentals of Periodontics- Wilson & KornmnPeriodontology 2000; vol 34; 2004British Dental Journal; vol 184: 1998.Internet.Presented by:Sanjeeva kumar reddy C.M