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 disease activity
No reliable criteria for identifying at risk individuals.
Inaccurate measurements

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

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. PERIOTEMP PROBE ADVANTAGES: DISADVANTAGES:
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 still not clear

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. Culturing -Advantages
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

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. Immuno Fluorescent Microscopy assays (IFA)
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.

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. DNA PROBE
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

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. Tissue breakdown products Inflammatory mediators and immune mediators.
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