ADVANCED DIAGNOSTIC AIDS

1. ADVANCED DIAGNOSTIC AIDS

2. CONTENTS • Limitations of conventional therapy • Advances in Clinical diagnosis Radiographic assessment Microbiologic Analysis Characterizing the Host Response

3. LIMITATIONS OF CONVENTIONAL DIAGNOSTIC AIDS Does not give information about: • Cause of the conditions • Patient’s susceptibility to disease • Progressing (or) Remission. • Response to therapy: positive (or) negative. • No reliable markers for diseaseactivity • No reliable criteria for identifying at risk individuals. • Inaccurate measurements

4. ADVANCED CLINICAL DIAGNOSIS • Clinical Diagnosis • Radiographic assessment • Microbiologic Analysis • Characterizing the Host Response

5. ADVANCES IN CLINICAL DIAGNOSIS Gingival bleeding Gingival Temperature Periodontal Probing

6. GINGIVAL BLEEDING Greenstein et al 1981 :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.

7. Gingival Bleeding.... Lang et al in a retrospective study reported that sites with BOP at several visits has higher probability of losing attachment than those that bleed on one visit (or) did not bleed. Lang et al demonstrated that any force greater than 0.25N might evoke bleeding in healthy sites with an intact periodontium.

8. Advantages Indicator of inflammatory lesion Indicator of disease activity Disadvantages Limited Predictability for disease progression Smoking may mask the bleeding. BLEEDIN ON PROBING

9. PERIODONTAL PROBING • Increased probing depth and loss of clinical attachment are path gnomonic of periodontitis • most widely used diagnostic tool for assessment of tissue destruction in periodontitis. • Readings of clinical pocket depth obtained with the periodontal probe do not normally coincide with the histologic pocket depth, since the probe normally penetrates the coronal level of the junctional epithelium.

10. CLASSIFICATION OF PROBES Generation I: Conventional probes Limitations: • Less sensitive, lack of Reproducibility • Measurements very subjective • Clinical pocket does not correlate with the histologic depth • The disparity between measurements also depends on • The probing technique • Probing force • Size of the probing • Angle of insertion of the probe • Precision of the probe calibration • Inflammation of tissues • Contour of teeth and roof surface.

11. Generation II:Pressure sensitive probes which have a standardized controlled insertion pressure. Generation III:Semi automated and automated probes, which are computerized. Florida probes, ‘Interprobe’ or the ‘Periprobe systems’. Generation IV: Probe aim at recording sequential probing positions along the gingival sulcus. Generation V: Probe would have an ultrasonic device attached to the fourth generation Probe for identifying attachment level without penetrating it.

12. SUBGINGIVAL TEMPERATURE Kung et al (1990), claim that these thermal problems are sensitive diagnostic devices , the periotemp probe (Abiodent, Inc. Davers, mass), detect pocket temperature differences of 0.1o C from a referenced for measuring early inflammatory changes in the gingival tissues. One commercially available systemsubgingival temperature.

13. ADVANTAGES: Need to treat become more easily recognized. Beneficial therapeutic response can be objectively demonstrated. Therapy can be more easily justified. Diagnosis becomes simple operative procedure. DISADVANTAGES: Less sensitive in the posterior region. However, the influence of pocket depth on temperature is stillnot clear PERIOTEMP PROBE

14. ADVANCES IN RADIOGRAPHIC ASSESSMENT • DIGITAL RADIOGRAPHY • SUBTRACTION RADIOGRAPHY • CADIA

15. DIGITAL RADIOGRAPHY • Digital radiography enables the use of computerized images, which can be stored, manipulated, and corrected for under and over exposures. • Digital radiography may yield almost equal image properties compared with conventional radiographs, but through digital storage and processing, diagnostic information can be enhanced (Hausmann 2000). • dose reduction : 1/3 to ½ than conventional radiographs

16. SUBTRACTION RADIOGRAPHY • Technique what facilities both qualitative and quantitative visualization of even minor density changes in the bone by removing the unchanged anatomic structures from the image • Improves the sensitivity and accuracy of evaluation • Relies on conversion of serial radiograph into digital images, this image can be superimposed and the resultant composite viewed on a video screen • Lighter areas- bone gain • Dark areas- bone loss • Requires paralleling technique to obtain a standardized geometry and accurate super imposable radiograph

17. SUBTRACTION RADIOGRAPHY….. ADVANTAGES • High degree of correlation between changes in the alveolar bone determined by subtraction radiography and serial changes in periodontal patients • Increased detectability of small osseous lesions • Used in longitudinal clinical studies DISADVANTAGES • Requires identical projection alignment during the exposure of sequential radiographs

18. COMPUTER ASSISTED DENSITOMETRIC IMAGE ANALYSIS SYSTEM • In this system, a video camera measures the light transmitted through a radiograph and the signals from the camera are converted into gray-scale images. The camera is interfaced with an image processor and a computer that allow the storage and mathematical manipulation of the images. • objective method for following alveolar bone density changes quantitatively over time • Has higher sensitivity and a high degree of reproducibility and accuracy.

19. ADVANCES IN MICROBIOLOGIC ANALYSIS Bacterial culturing Direct microscopy Enzymatic methods Assays based on molecular biology techniques

20. MICROBIOLOGIC ANALYSIS • Helps in diagnosis of the various forms of periodontal disease, • 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. • Helps to monitor periodontal therapy directed at the suppression (or) eradication of periodontopathic microorganisms.

21. BACTERIAL CULTURING Plaque samples are collected and are cultured in • Selective Media • Non-Selective Media

22. Can obtain relative and absolute counts of cultured species Only in vitro method for Antibiotic susceptibility Has the potential to support the diagnosis of various forms of periodontal disease Serve as indicators of disease initiation and progression Culturing -Advantages

23. Disadvantages • Strict sampling • Collecting an adequate sample • Transport conditions anaerobic & fast • Treponema & Bacteroids : Difficult to culture

24. Direct Microscopy Direct Microscopy • Dark field • Phase contrast microscopy

25. Immuno Diagnostic Methods , • Direct and indirect immuno fluorescent microcopy assays (IFA), • Flow cytometry, • Enzyme-linked immunosorbent assay (ELISA) • Latex agglutination • Membrane assay.

26. Direct IFA employs both monoclonal and polyclonal antibodies conjugated to a fluorescent marker that binds with the bacterial antigen to form a fluorescent immunocomplex detectable under a microscope. Immuno Fluorescent Microscopy assays (IFA)

27. Indirect IFA Employs a secondary fluorescence conjugated antibody that reacts with the primary antigen-antibody complex.

28. Flow Cytometry (or) Cytofluorography • It is for the rapid identification of oral bacteria involves labeling bacterial cells from a patient plaque sample with both species – specific antibody and a second fluorescent – 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. • Disadvantage: The sophistication and cost involved in this procedure precludes its wide usage.

29. ELISA:Enzyme Linked Immunosorbant assay • It is similar in principle to other radio immunoassays, but an enzymatically derived color reaction is substituted as the label in place of the radio isotope. • INTENSITY OF COLOR depends on the Concentration Of The Antigen and is usually read photo metrically for optimal quantization. USES • Primarily to detect serum antibodies to periodontal pathogens • also been used in research studies to quantify specific pathogens in sub gingival samples using specific monoclonal antibodies

30. Latex agglutination: • It is a very simple immunological assay Based On The Binding Of Protein To Latex. • Latex beads are coated with the species specific antibody -- 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. • ADVANTAGE: Because of their simplicity and rapidity, these assays have great potential for chair side detection of periodontal pathogens.

31. Membrane Assay It involves linkage between the antigen and a membrane bound antibody to form an immunocomplex that is later revealed through a colorimetric reaction. Evalusite has been designed to detect A.actinomycetemcomitans, P.gingivalis, and p.intermedia

32. Enzymatic methods of bacterial identification: BANA (N-Benzoyl-DL-arginine-2 napthylamide) assay PRINCIPLE: Organisms – species sharing a common enzymatic profile (trypsin like enzyme) Activity measured by the hydrolysis of the colorless substrate BANA Produces a blue black color Identity proportional to the total amount of the four orgs. (B.forsythis, p.gingivalis, t.denticola, and capnocytophaga)

33. Perio scan(Bana) • Chair side diagnostic kit developed using this reaction • Hydrolysis – releases chromophore napthylamide • Turns orange red when a fast garnet is added to the solution • Utility – uncertain due to its low reliablity to predict clinical assessment of disease progression.

34. Perio scan(Bana) Advantages: Serve as a marker of disease activity. Loesche et al showed that shallow pockets exhibited only 10% positive BANA reactions, where as deep pockets (7mm) exhibited 80% to 90% positive BANA reaction. Beck et al used the BANA test as a risk indicator for periodontal attachment loss. BANA Test – Difficulties: • If may be positive in clinically healthy sites and remains to be proven whether it can detect sites undergoing periodontal destruction. • It only identify a very limited number of pathogens, its negative result does not rule out the presence of other periodontal pathogens.

35. Molecular Biology Techniques PRINCIPLE: reside in the analysis of DNA, RNA and Structure or Function of Protein Requires DNA Fragments that recognize complementary-specific bacterial DNA sequences from target organism. Requirement: ability to extract bacterial DNA from plaque sample DNA sequence of the target periodontal pathogens.

36. DNA PROBE Deoxyribonucleic acid probe Nucleic acid Probes: • Deoxy Ribonucleic Acid (DNA) probes entail segments of single – stranded nucleic acid, labeled with an enzyme (or) radioisotope, that on locate and bind to their complementary nucleic acid sequences with low cross – reactivity to non target organisms. • DNA probe may target whole genomic DNA (or) individuals genes.

37. Whole genomic probes are more likely to cross react with non target microorganisms due to the presence of homologous sequences between different bacterial species. 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 DNA PROBE

38. DNA PROBE Advantages: • They detect rapidly multiple bacteria, like A.actinomycetemcomitans, P.gingivalis, P.intermedia, C.rectus, E.corrodens, Fusobacterium nucleatum and T.denticola. • The probes are able to detect as few as 102 to 104 bacteria, and • Sensitivity and specificity are not affected by the presence of unrelated bacteria.

39. DNA PROBE • Commercially available kits: • DMDx TEST KIT • OMNIGENE • BTD (Biotechnica Diagnostics, inc)

40. Polymerase chain reaction (PCR): • Developed in 1985 • Polymerize chain reaction (PCR) involves amplification of a region of DNA flanked by a selected primer specific for the target species. • The presence of the specific amplification product indicates the presence of the target microorganism • Best detection limits, as 5-10 cells and shows no cross reactivity. • 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.

41. Polymerase chain reaction (PCR): • Reverse Transcriptase PCR(RT PCR) RNA-- (rev transcriptase)—DNA—Normal PCR • End Point PCR- Quantities assessment Disadvantage: amt. of PCR product shows week correlation with initial quality • Real Time PCR: Quantitive assessment High degree of specifity, sensitivity, & reproducibility Disadvantage: Very expensive

42. PCR Advantages: • Little amount of DNA sample is sufficient and can be used to detect pathogens that are slow (or) dangerous to grow. • Future of Microbial Analysis: • Patients who do not respond favourablly to conventional mechanical therapy. Disadvantage • Small plaque sample- if doesn’t contain organism. • Sub gingival plaque may contain enzymes that can alter the amplification process. LIMITATION is that information obtained is relevant to the site sampled and hence may not be representative of the microflora of the entire dentition.

43. ADVANCES IN CHARECTERISING THE HIST RESPONSE

44. BIOCHEMICAL DIAGNOSTIC AIDS: • Assessment of the host response refers to the study of mediators, by immunologic (or) biochemical methods. 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. .

45. BIOCHEMICAL DIAGNOSTIC AIDS: Source of samples: • Potential sample sources include Saliva, gingival crevicular fluid (GCF), gingival crevicular cells, blood serum, blood cells, and urine. analysis of urine shows little promise. • Most efforts to date have been based on the use of components of GCF and, to a lesser extent, Saliva and blood (Page 1992).

46. Studies have demonstrated : Increased amounts of GCF flow in gingivitis. • more than 40 components of GCF have been studied. Divided into four main groups • Host derived enzymes, • Tissue breakdown products • Inflammatory mediators and immune mediators. • Assessment of susceptible host for markers in Peripheral blood.

47. Chairside Procedures • Periogard- Aspartate aminotransferase (AST) • Perocheck- Elastase • Evalusite- ELISA • Test Stick- MMP 8 (Collagenase) • BANA Test- (N-Benzoyl-DL-arginine-2 napthylamide) assay • Identification of Red complex bacteria (pg, Trep d, taneralla forsytha) • Periotest- Mobility • Perioscopy- visualization, detection

48. Conclusion

49. Thank You