ADVANCED DIAGNOSTIC TECHNIQUES LIMITATIONS OF CONVENTIONAL PERIODONTAL DIAGNOSIS Periodontal diseases are prevalent human diseases defined by the signs and symptoms of gingival inflammation and periodontal tissue destruction. These diseases are conventionally diagnosed by clinical evaluation of the signs of inflammation in the gingiva without periodontal tissue destruction (gingivitis) or by the presence of both inflammation and tissue destruction (periodontitis). The traditional clinical diagnosis is made by measuring either the loss of connective tissue attachment to the root surface (clinical attachment loss) or the loss of alveolar bone (radiographic bone loss). However, this evaluation cannot reliably identify sites with ongoing periodontal destruction and does not provide any information on the cause of the condition, on the patients susceptibility to disease, whether the disease is progressing, whether it is in remission, or whether the response to therapy will be positive or negative. The current view of the natural history of destructive periodontal disease is that disease susceptibility is related to the whole person rather than the local site. This means that a persons susceptibility and host defense mechanisms are generalized. However, the disease process itself is considered to be site specific and has a multifactorial origin in which periodontal pathogens, host response, and genetic, systemic, and behavioral risk factors interplay to develop the disease process. In light of this information, consideration 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. This topic systematically reviews the advances made in the use of these parameters. Advances in clinical diagnosis Gingival temperature Periodontal probing Dental endoscope Advances in microbiologic analysis Bacterial culturing Direct microscopy Immunodiagnostic methods Enzymatic methods of bacterial identification Deoxyribonucleic acid probe technology[DNA probe] Restriction Endonuclease analysis Polymerase chain reaction[PCR]

Advances in characterizing host response Source of samples Inflammatory mediators and products Host derived enzymes Tissue breakdown products Assessment of susceptible host for markers in peripheral blood. Advances in radiographic assessment Digital radiography Substraction radiography Computer assisted densitometric analysis Computed tomography Magnetic resonance imaging Nuclear medicine scans SUBGINGIVAL BLEEDING Clinical evaluation of the degree of gingival inflammation includes assessment of the redness and swelling of the gingival along with assessment of gingival bleeding. Gingival bleeding is related to the persistent presence of plaque on the teeth and is regarded as a sign of the associated inflammatory response. The use of gingival bleeding as an indicator of inflammation has the clinical advantage of being more objective, because color changes require a subjective estimation. It has also been shown that gingival bleeding is a good indicator of the presence of an inflammatory lesion in the connective tissue at the base of the sulcus and that the severity of bleeding increases with an increase in size of the inflammatory infiltrate. Therefore, clinicians tend to evaluate gingivitis by gingival bleeding alone, with the use of a periodontal probe or signs of both inflammation and bleeding.

Limitations-Lang et al. demonstrated that any force greater than 0.25 N may evokebleeding in healthy sites with an intact periodontium. Although bleeding on probing may have a limited predictive value for disease progression, its absence indicates periodontal stability with high probability. SUBGINGIVAL TEMPERATURE Local physical changes such as temperature elevation can be used for prediction of metabolic events occurring in periodontal tissues. Early studies by Kung et al 1990 linked subgingival temperature with periodontal pocketing. Maxillary periodontal sites are hotter than mandibular periodontal sites and posterior sites are hotter than anterior sites [Haffajee et al 1992]. There is a positive correlation between elevated subgingival temperature and

-Severity of disease -Degree of gingival inflammation -Presence of putative pathogens. Subgingival temperature, like other signs of inflammation has good specificity but poor sensitivity when considered alone as a marker for progressive periodontitis. PERIODONTAL PROBING -Widely used diagnostic tool for measurement of CAL loss. -Increased pocket depth and loss of clinical attachment level is pathogneumonic sign for periodontitis. -Clinical pocket depth does not coincide with the histologic pocket depth.-because probe penetrates the coronal level of the junctional epithelium -If the tissues are also inflamed then the tissue offer's less resistance to probe penetration false reading is obtained. The difference in measurements depends upon the Probing technique Probing force Size of the probe Angle of insertion of the probe Precision of the probe calibration Forces up to 30 gm the tip of the probe seems to be in the junctional epithelium Forces up to 50 gm - necessary to diagnose periodontal osseous defects Various generations of probe development: Generation I: conventional probes. Generation II: Pressure sensitive. Generation III: Computerized. Generation IV & V probes is currently under development. Generation IV probes aim at recording sequential probing positions along the gingival sulcus. Generation V: would have an ultrasonic device attached to the fourth generation probe for identifying attachment level without penetrating it. Probing measurements can be affected by incorrect angulation /interference with the calculus/presence of overhanging restorations Measurements can also be affected by force applied. Can be overcome by using automated probes.

Currently used automated probes Florida probes( GIBBS et al) Features Constant probing force Precise electronic measurement Computer storage of data Precise early detection of disease Clear graphic charting Components Probe hand pieces Digital read out 3- Pedal foot switch Computer interface FEATURES OF HAND PIECE Probe tips are manufactured by micro polishing technique Constant force measurements in 0.2 mm Probe tips are 0.4 millimeter in diameter made of implant grade titanium Can be steam sterilized in a standard autoclave

Three Pedal foot switch Solo operator may enter all data Eliminates error in visual reading Less contamination

Computer interface Pocket depth & bleeding sites called out automatically using sound effects from computer Color coded digital read outs Educates and motivates patient

Two models Stent model Has 1 millimeter metal collar that rests on a prepared ledge on a prefabricated vaccuoform stent. Disk model Has 1 millimeter disk which rests on the occlusal surface/ incisal edge of tooth. The disk is placed on the occlusal/incisal surface while measuring the probing a pocket.

Jeffcoat probes [Foster Miller probes] Automatically extends & retracts under controlled force Rapid change in acceleration when C-E Junction is crossed Working end of probe has Michigan O probe markings Internal moving tip Teflon coated stainless steel wire Circular in cross section & 0.15 millimeter in diameter Probe motion provided by custom low friction pneumatic cylinder Toronto probes Uses occlusal incisal surface as reference Sulcus probed with 0.5 millimeter nickel - titanium wire under air pressure Mercury tilt sensor limits angulation within 30o

INTER PROBE ( Goodson & Kondon 1988 ) Electronic probe using an optical encoder transduction element BIREK PROBE Works under constant air pressure Uses occlusal surface as reference THE DENTAL ENDOSCOPE A dental endoscope has been introduced recently for use subgingivally in the diagnosis and treatment of periodontal disease. Produced by Dental View, Inc.- called as Perioscopy system. It consists of 0.99mm diameter reusable fibroptic endoscope over which is fitted a disposable, sterile sheath. The fibroptic endoscope fits on to the periodontal probes and ultrasonic instruments that have been designed to accept it. The sheath delivers water irrigation that flushes the pocket while the endoscope is in use and keeps the field clear. The fibroptic endoscope attaches to a Medical grade Charged Coupled Device [CCD], Video camera and Light source that produces a image on a flat panel video monitor for viewing during subgingival exploration and instrumentation. Magnification ranges from 24X to 46X, enabling visualization of even minute deposits of plaque and calculus.

Uses: Allows clear visualization of deep subgingival pockets and furcations. Enables operator to detect the presence and location of subgingival deposits and guides the operator in their removal. Using this device it is possible to achieve levels of root debridement and cleanliness that are much more difficult to produce without it. Can also be used to evaluate subgingivally For caries, Defective restorations, Root fractures and Resorption. These are diamond abrasive files. These are just two of the special instruments used to remove calculus that is adherent to the root surface. This is another example of a special root planing instrument. It is a slotted file. The end of the instrument is about 1.5 mm in diameter. It reaches areas of the root surface that conventional root planing instruments can not reach.

Advances in microbiologic analysisMICROBIOLOGIC ANALYSIS Since subgingival oral bacteria are the main initiating agents in the development of periodontal disease, it makes sense to look for specific bacteria in the patients with periodontal disease. Several methods have been employed for the detection of putative periodontal pathogens in subgingival samples. Some of these methods are used strictly for research purposes while other methods are adapted for clinical use. All these methods require subgingival plaque as sample. The essential element in periodontal microbiology is: Selection of proper sample site. Collecting adequate sample size. Collection of these samples become difficult in patients infected with organisms that are unevenly distributed. Mombelli et al reported that four individual subgingival specimens each from the deepest periodontal pocket from each quadrant should be pooled to detect Porphyromonas gingivalis. Uses include: These tests have the potential To support the diagnosis of various forms of periodontal diseases. To serve as indicators of disease initiation and progression [i.e., disease activity] To determine which periodontal sites are at higher risk for active destruction.

To monitor periodontal therapy directed at periodontopathogenic microorganisms.

the suppression or eradication of

Predictive treatment model [1989] By Kornman and Newman. A method has been proposed that uses a combination of clinical and microbiologic parameters to predictably recommend specific therapy -This is termed as Predictive treatment model. They used this model in the treatment of refractory periodontitis patients. DIRECT MICROSCOPY Dark field / Phase contrast microscopy: It is an alternative for culture methods on the basis of its ability to directly or rapidly assess the morphology and motility of bacteria in a plaque sample. Uses: Used to indicate periodontal disease status and to structure maintenance programs. Demerits: Most of the putative periodontopathogenic organisms like A.actinomycetamcomitans, P.gingivalis, B.forsythus,E.corrodens and Eubacterium species are non motile. Hence this technique is unable to identify these species. Unable to differentiate among various species of Treponema. Hence dark field microscopy seems to be unlikely candidate as a diagnostic test of destructive periodontal disease.

BACTERIAL CULTURING Considered as the reference method/gold standard when determing the performance of new microbial diagnostic methods. Historically has been used widely and aimed at characterizing the composition of the subgingival microflora. Generally, plaque samples are cultivated anaerobically and by using selective media, together with several biochemical and physical tests the different putative organisms can be identified. ADVANTAGES: We can obtain relative and absolute counts of cultured species. Moreover it is the only invitro assessment for antibiotic susceptibility of the microbes SHORT COMINGS Culture methods can grow only live bacteria, therefore strict sampling and transport condition are essential. Some of the putative periodontopathogens such as Treponemas sps and Bacteroides forsythus are fastidious and are difficult to culture. The sensitivity is very low, since the detection limits for selective and non selective media average 103 to 104 bacteria leading to undetection of low numbers of specific pathogens in a pocket. Most importantly culture require sophisticated equipment and experienced personnel Time consuming and expensive. IMMUNO DIAGNOSTIC METHODS Immunologic assays employ antibiotics that recognize specific bacterial antigens to detect target microorganisms. This reaction can be revealed using different procedures like: Direct and indirect immunoflouresent microscopy assays Flow cytometry Enzyme linked immunosorbent assay Membrane assay Latex agglutination. IMMUNOFLOURESCENT MICROSCOPY Direct: Employs both monoclonal and polyclonal antibodies conjugated to a fluorescein marker that binds with bacterial antigen to form a fluorescent immune complex, which is detectable under a microscope. Indirect: Employs a secondary -fluorescein conjugated antibody that reacts with the primary antigen antibody complex. Both these are able to detect the pathogens and quantify the percentage of the pathogens directly from a plaque smear. Has been widely used to detect A.a, P.gingivalis.

This technique is comparable to bacterial culture in its ability to identify these pathogens in subgingival dental plaque samples[Zambon et al ]. It does not require viable bacterial cells like bacterial culturing. Sensitivity of these assays 82%-100% for detection of A.a; 91-100% P.gingivalis. Specificity 88-92% for A.a; 87-89% for P.gingivalis. CYTOFLUOROGRAPHY Flow cytometry. This involves rapid identification of oral bacteria. This method involves labeling bacterial cells from a patient plaque sample with both species specific antibody and a second fluorescein conjugated antibody. Then, this 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. After incubation the cells are passed through a focused laser beam. The cells scatter the light at low and wide angles, and the fluorescent emission can be measured by appropriate detectors. Disadvantages: Expensive Time consuming ELISA Enzyme linked immunosorbent assay Is similar in principle to other radioimmunoassays but it is enzymatically derived in place of radio isotope. ELISA detects either antigens or antibodies. Antigens are incubated in wells in a plastic plate to allow absorption and binding of the material. After washing to remove the unbound antigens, samples containing suspected antigen is incubated with known antibodies to bind to the antigen on the surface of the well After washing, antisera to the immunoglobulin conjugated to either alkaline phosphatase or horse shoe peroxidase are incubated in the wells. A positive reaction is visualized as solution changes from colorless to colored The intensity of color depends on the concentration of the antigens and is usually read photometrically for optimal quantification.

Uses: Used primarily to detect serum antibodies to periodontopathogens. It is used in research studies to quantify specific pathogens in subgingival samples using specific monoclonal antibodies. Commercially available as, Evalusite[Kodak] using antibodies to detect antigens for P.gingivalis, P.intermedia A.a, Simple chair side kit. LATEX AGGLUTINATION It is a simple immunological assay based on the binding of protein to latex. Latex beads are coated with species specific antibody and when these beads come in contact with the microbial cell surface antigens or antigen extracts cross linking occurs. Agglutination or clumping is visible in 2-5 minutes. Two types: Direct Inhibition Direct assay: It is a common latex agglutination test for bacteria. The antibody is bound to latex, when a suspension of plaque sample is mixed with sensitized latex and gently agitated for 3-5 minutes resulting in agglutination or clumpingInhibition assay: Based on the principle that inhibiting the expected agglutination reaction between known antigen and known antibody as a result of competition. Advantages: Simplicity and rapidity. Used as chair side detection of periodontal pathogens. Particle concentration fluorescence immuno assays[PCIFA] Uses bacterial cells as a solid phase together with LPS specific monoclonal antibodies. The resulting assays exhibits 97%-100% of sensitivity and 57% to 97% specificity. Selection limits of 104 cells. Disadvantages of immunological assays: Although used extensively, lack clinical validation since most of them are not available commercially. Cross reactivity leading to detection of false positives- major problem -when polyclonal antibodies are used. False negatives occur when using monoclonal antibodies when compared with culture.

Can identify dead target cells, thus not requiring stringent sampling and transport methodology. Most of these, Cannot be used to determine antibiotic susceptibility Provide only quantitative or semi quantitative estimate of target organisms. Show poorer detection limits than nucleic acid probes/PCR assays. ENZYMATIC METHODS OF BACTERIAL IDENTIFICATION:BANA TEST Based on the activity of trypsin like enzyme with N benzoyl dl arginine 2 napthylamide. The activity of this enzyme can be measured with the hydrolysis of the colorless substrate BANA. When hydrolysis takes place, it releases the chromophore, beta napthylamide, which turns orange red when a drop of fast garnet is added to the solution. B.forsythus,P.gingivalis,T.denticola,Capnocyto-phaga sp.,- all share a common enzymatic profile, since all have an common trypsin like enzyme. Diagnostic kits are available PERIOSCAN. Loesche et al : Studied the reaction of BANA with subgingival plaque samples in shallow pockets and deep pockets to detect presence of these pathogens and thus serve as marker of disease activity Using pocket depth as measure of periodontal morbitity he showed that Shallow pockets less than or equal to 5mm exihibited only 10% of positive BANA reactions. Deep pockets greater than or equal to 7 mm exhibited 80 90 % of positive BANA reactions. Beck et al: Used BANA test as an indicator for periodontal attachment loss. Taken collectively , positive BANA findings are a T.denticola/P.gingivalis or both are present at sample sites.

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Difficulties: It may be positive at clinically healthy sites and remains to be proven whether it can detect sites undergoing periodontal destruction. It only detects a very limited number of pathogens, its negative result does not rule out the presence of other periodontal pathogens. DEOXYRIBONUCLEIC ACID PROBE TECHNOLOGY NUCLEIC ACID PROBES: DNA probes consists of single stranded nucleic acid, labeled with an enzyme or radio isotope that can locate and bind to their complimentary nucleic acid sequences with cross reactivity to non target microorganisms. DNA probe may target whole genomic DNA or individual genes.

Whole genomic probes are more likely to cross react with non target microorganisms due to the presence of homologous sequences between different bacterial species. However, specific genes, such a 16s RNA genes, contain signature sequences limited to organisms of same species. These oligonucleotides probes display limited or no cross reactivity with non target microbes. How it is prepared? To prepare the probe, specific pathogens used as marker organisms are lysed to remove their DNA. Their double helix is denatured, creating single strands that are individually labeled with a radioactive isotope. Subsequently when a plaque sample is sent for analysis, it undergoes lysis and denaturation. Single strands are chemically treated, attached to a special filter paper and then exposed to DNA library. If the complimentary base pairs hybridize [cross link], the radio labeled strands will also be fixed to the filter paper. After the filter is washed to remove any unhybridized strands, it is covered with a radiographic plate. The radioactive labels create spots on the film, which are read with a densitometer. The darkness and size of the spots indicate the concentration of the organism present in the given plaque samples. Advantages : Assay can rapidly test for multiple bacteria, like A.a., P.gingivalis, B.intermedius, C.rectus, E.corrodens, Fusobacterium nucleatum and T.denticola. Can detect as few as 102 104 bacteria. Sensitivity and specificity are not affected by the presence of untreated bacteria in mixed culture samples. Commercially available kits: -DMDX TEST KIT -OMNIGENE -BTD[Biotechnica Diagnostics., Inc] RESTRICTION ENDONUCLEASE ANALYSIS Restriction endonucleases recognize the double stranded DNA at specific base pair sequences. The DNA fragments thus obtained, are separated by electrophoresis, Then stained with ethidium bromide and visualized with ultraviolet light. The genetic heterogenicity and homogenecity of strains can be evaluated by comparing the number and size [electrophoretic pattern]of the DNA fragments obtained. These DNA fragments patterns constitute a specific finger print to characterize each strain. Uses:

REA is thus powerful tool for determining the distribution of a specific pathogenic strain through out a population. Also used in molecular genetic analysis of diverse oral bacteria like A.a., P.g.,P.i., E.c.,F.n.,T.d., Has been very useful in studying the transmission patterns of putative periodontal pathogens among family members. POLYMERASE CHAIN REACTION PCR involves a reiterate amplification of a region of DNA flanked by selected primer specific for the target species. The presence of specific amplification product indicates the presence of target microorganisms. Different bacterial species may be detected simultaneously by multiplex PCR in which several distinct primer pairs each specific for a given target microorganisms are employed in a given single tube amplification. Advantages: Demonstrates best detection limits,as few as five to ten cells. Shows no cross reactivity under optimized amplification conditions Easy to perform. Drawbacks: Most PCR assays use relatively small aliquots for amplification process. If this small quantity of plaque sample does not contain the targeted microorganisms the assay will not detect it. Moreover, subgingival plaque, contain many enzymes that can alter the amplification process. Recently, Fijise et al developed a quantitative PCR method. ADVANCES IN CHARACTERIZING THE HOST RESPONSE ADVANCES IN CHARACTERIZING THE HOST RESPONSE Assessment of host response refers to 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 or like local release of inflammatory mediators, host derived enzymes or tissue breakdown products. Diagnostic tests based on these systems are usually non invasive. Source of samples: Saliva GCF Blood serum Blood cells Urine

Most of the tests are based on components of GCF and to a lesser extent, saliva and blood. ANALYSIS OF URINE Shows little promise Except for its use in differential diagnosis of tooth loss related to hypophosphatasia in young children. Here presence of phosphoethanolamine in the urine is diagnostic of the disease. ANALYSIS OF SALIVA: Easily collected Contain both locally and systemically derived markers of periodontal disease. Collected from parotid, submandibular and sublingual glands Whole saliva consists of mixture of oral fluids, including secretions from Major/minor salivary glands Constituents of non salivary origin GCF, bacteria, bacterial products, desquamated cells, expectorated bronchial secretions. Obtained with or without stimulation. Proposed diagnostic markers in saliva include Proteins and enzymes of host origin Phenotypic markers Host cells Hormones-cortisol Bacteria and products Volatile compounds and ions. Biochemical markers in saliva: Epithelial keratins in GCF: It has been suggested that phenotypic markers for junctional, sulcular and oral epithelial cells might eventually be used as indicators of periodontal diseases. The keratin concentration in GCF was higher at sites exhibiting signs of gingivitis and periodontitis compared with healthy sites[Mchanghlin et al 1996] Hence keratin concentration may serve as marker of gingival inflammation. Salivary ions: Calcium is the ion that has been most widely studied as a potential marker for periodontal disease in saliva. An elevated calcium concentration in saliva was characteristic of patients[Sewon et al 1995]with periodontitis. Serum markers in saliva cortisol Recent studies have suggested that emotional stress is a risk factor for periodontitis.

One mechanism proposed - serum cortisol levels associated with emotional stress exert a strong inhibitory effect on the inflammatory process and immune response.[Genco et al 1998]. ANALYSIS OF GCF Most of the diagnostic tests in Periodontics have the limitation that cannot differentiate between active and inactive sites nor can identify susceptible individuals. To overcome this difficulty, the components in GCF have been studied over the years. More than 40 GCF components have been studied. They can be divided into 3 main groups. Host derived enzymes Tissue breakdown products Inflammatory mediators. Collection of GCF: Paper strips Micro capillary tubes Micropipettes Micro syringes Plastic strips Widely used paper strips. How is it collected? These strips are placed in the gingival sulcus for standard period of time until the filter paper is saturated. Fluid volume collected from the strips are quantified by using Periotron This electronic device measures the change in the capacitance across the wetted strip and this change is converted to the volume of GCF. Periotron 8000 achieves the easiest and quickest measurement. Research on GCF has focused on the search for biochemical markers for the disease progression, many of which are discussed below: INFLAMMATORY PRODUCTS AND MEDIATORS Cytokines are potent local mediators of inflammation that are produced by variety of cells. The common inflammatory mediators present in the GCF are investigated as potential diagnostic markers: Prostoglandin E2 Tumor necrosis factor Interleukin 1 and 1 Interleukin 6 and 8. Prostaglandin E2: Product of cyclooxygenase pathway of the arachidonic acid metabolism.

Potent mediator of inflammation and induces bone resorption. In cases of untreated periodontitis, the concentration of Prostaglandin E2 in GCF is increased during active phases of periodontal destruction. Interleukin 1, 1, 6,8, TNF alpha: Produced by macrophages at diseased sites. Potent immune regulatory molecules with a variety of biologic effectsMatrix metalloproteinase stimulation Bone resorption. Cross sectional studies have shown good correlation with disease status and severity but not disease progression. HOST DERIVED ENZYMES Various enzymes are released from the host cells during the initiation and progression of periodontal disease. These enzymes are released from, dead and dying cells from periodontium PMN neutrophils others, derived from inflammatory, epithelial and connective tissue cells at the affected sites. The enzymes that received most attention as periodontal destruction are: Aspartate aminotransferase Alkaline phosphatase Beta glucronidase Elastase Cathepsin Aspartate aminotransferase: Enzyme derived from variety of cells through out the body including the heart and the liver Elevated AST in GCF mainly from sites with severe gingival inflammation and sites with recent history of progressive attachment loss. Test involves, Collection GCF with filter paper strip which is then placed in tromethamine hydrochloride buffer. Substrate reaction mixture containing L-aspartate and - ketogluterarate acids are added and allowed to react for 10 minutes. In the presence of AST, The aspartate and -glutarate are catalysed to oxalo-acetate and glutamate. The addition of a dye such as fast red results in a color product and the intensity is proportional to the AST activity in GCF sample.

Disadvantage: Its inability to discriminate between sites with severe inflammation but with no attachment loss from sites that are losing attachment. Commercially available kits: PeriogardTM- periodontal tissue monitor system [ Xytronyx Inc, San Diego,LA, USA] Pocket watchTM [Steri Oss Inc., Yorba, Linda, CA, USA] Alkaline phosphatase: Found in many cells of periodontium including osteoblasts, fibroblasts and neutrophils. Cross sectional studies show that higher levels are seen in GCF of diseased sites than from healthy sites. Only one longitudinal study has associated whole mouth alkaline phosphatase levels with the progression of periodontitis but this study has not been reproduced[Binder et al 1987] Beta glucronidase: Found in primary [azrophilic] granules of neutrophils. Cross sectional data show that elevated beta glucronidase in diseased sites. Two longitudinal studies have shown that concentration of this enzymes have predictive value in identifying patients at higher risk for losing attachment[Lamster et al 1988]. Commercial available kits: Is being developed by ABBOTT laboratories, North Chicago, USA. Elastase: It is a serine protease stored in the primary granules of neutrophils. Cross sectional studies showed that higher levels of elastase are seen in sites with periodontitis than with gingivitis/healthy sites. Longitudinal studies has shown that some predictive value for disease progression with elastase can be obtained in a short term evaluation of an untreated population, but it remains if its validity can be applied to a treated population in a maintenance program. Commercially available kits: Periocheck: Measures presence of nonspecific neutral protease activity. Prognostik[Dentsply, USA] Presence of serine proteases, elastase in GCF. Cathepsin: These are a group of acidic lysosomal enzymes that play an important role in intra cellular protein degradation. Although they have been shown correlation with disease severity and significant decrease after periodontal therapy, they have not been evaluated longitudinally as markers of disease progression.[Chen et al 1998]

Tissue breakdown products: One of the major feature of periodontitis is the destruction of collagen and extracellular matrices. Analysis shows that sites with periodontitis shows elevated levels of Hydroxy proline from collagen breakdown Gycosaminoglycans from matrix degradation. Osteocalcin and type I collagen peptides These are bone and connective tissue proteins that have been correlated with the progression of alveolar bone loss induced in beagle dogs [Giannobile et al 1995]. Both markers showed high predictive value and now need to be extended to longitudinal studies in humans. Assessment of susceptible host using markers in peripheral blood: The contents of circulating blood can be used to diagnose periodontal disease has been considered for several years. It will identify patients at risk rather identify tooth/teeth sites that are with increased disease activity.[Fine and Mandrel 1986,Johnson 1989]. Three constituents of peripheral blood have been thoroughly pursued as assessment of susceptibility: Polymorphonuclear leukocyte function Estimating antibody levels to bacterial antigens Monocytes responsiveness to bacterial LPS. Polymorphonuclear leukocyte [PMNS] Studies by Lavine et al 1976 showed an essential role for the neutrophil in the pathogenesis of periodontal disease. Defects in the neutrophil functions such as chemotaxis, phagocytosis has been clearly associated with localized and generalized aggressive periodontitis and also with systemic conditions like Downs syndrome, agranulocytosis, PLS,DM, etc., Genco et al in 1986 proposed that transient neutrophil abnormalities in adults may lead to periods of increased disease activity. It seems that measuring neutrophil function should be used for screening individuals at high risk for active periodontal disease progression. Since laboratory assessment is very expensive and time consuming, this diagnostic test not yet is applicable for everyday practice. Antibody titers: For many years it has been suggested that circulating blood contains antibody titers to periodontal pocket bacteria. Caton et al 1989 reported that elevation in serum antibody to plaque bacteria is associated with increasing severity of periodontal disease.

Ebersole in 1987 in his study, though incomplete, suggested that most patients with periodontal disease exhibit significant elevated antibody responses to suspected periodontopathogens. Thus this antibody measurement was reflection of host response to an infection associated with episodes of active periodontal disease progression. Monocytes responsiveness to LPS: Garrison and Nichols in 1989 reported an interesting test on patient susceptibility or resistance to periodontal disease based on possible altered host response - monocyte responsiveness. They hypothesized that susceptibility to periodontal destruction could be related to an increased host response to gram-negative bacteria. Subsequently, they compared susceptible patients with generalized severe adult periodontitis to patients resistant to periodontitis. Patients who were classified as susceptible demonstrated 2-3 fold greater responsiveness of blood monocyte to LPS [i.e., as measured by PGE2 release] than did patients classified as resistant. Interestingly, susceptible patients with severe disease had greater monocyte responsiveness, regardless of their treatment stage. This test needs further research, since susceptibility may also have a genetic basis. Since these tests offer information at the patient level, offering screening of risk patients but have a limitation in relating information on status of particular sites within patients.