Advanced diagnostic aids in periodontics

1. Advanced Periodontal
Diagnostic Techniques
(As an adjunct to conventional techniques)

2. Content of discussion
 Introduction
 Limitations of conventional periodontal diagnosis
 Advances in Clinical diagnosis
 Advances in Radiographic assessment
 Advances in Microbiologic analysis
 Advances in characterizing the Host response
 Conclusion and future scopes

3. Introduction
Definition of DIAGNOSIS (Dx)* :
Diagnosis is defined as; correct determination,
discriminative estimation and logical appraisal of
conditions found during examination as evidenced by
distinctive marks, signs and characteristics of diseases
*Glossary of Periodontal terms

4. Current conventional techniques
 Clinical diagnosis is made by measuring either clinical attachment
loss (CAL) or radiographically by loss of alveolar bone
 This kind of evaluation identify and quantify current clinical signs of
inflammation ,
 Provides historical evidence of damage with its extent and severity

5. Limitations !!!
 Does not provide cause of the condition
 No info. on patient’s susceptibility to the disease
 Cannot identify sites with ongoing periodontal destruction or
sites in remission
 Cannot differentiate whether response to therapy is positive or
negative

6. Advanced periodontal diagnostic techniques
Advances in Clinical diagnosis
Advances in Radiographic Assessment
Advances in Microbiologic Analysis
Advances in Characterizing the Host Response

7. Advances in
Clinical Diagnosis

8. 1. Gingival temperature
 Thermal probes are sensitive diagnostic devices for measuring
early inflammatory changes in gingival tissue.
 Subgingival temperature at diseased sites is increased as
compared to normal healthy sites
 Commercially available system PerioTemp probe enables the
calculation of temperature differential (with sensitivity of 0.10C)
between the probed pocket and subgingival temperature

9.  Possible explanation for ↑ temperature with increasing
probing depth is an increase in cellular and molecular activity
caused by increased periodontal inflammation
 Haffajee et al. (1992): found that elevated subgingival site
temperature is related to attachment loss in shallow pockets
and elevated proportions of Pg, Pi, Tf, Aa.

10. 2. Periodontal probing
 Most widely used diagnostic tool
 Probing depth is measured from the free
gingival margin to the depth of the
probeble crevice.
 Longitudinal measurement of CAL or
probing depth is a ‘gold standard’ for
recording changes in periodontal status

11. Limitation of conventional probing
 Lack of sensitivity and reproducibility.
 Disparity between measurement depends on:
probing technique, probing force, angle of insertion of probe,
size of probe, precision of calibration, presence of inflammation.
 Readings of clinical pocket depth measured with probe does not
coincide with the histologic pocket depth.
 All these variable contribute to the large standard deviations
(0.5-1.3 mm) in clinical probing results

12. Classification of periodontal probes
depending on generation

13. 2.Second generation probes: (Constant force probes)
 force to probe pocket: 30g
 force to probe osseous defect: 50g
 Introduction of constant force or
pressure sensitive probes allowed for improved
standardization of probing.
e.g.: Pressure sensitive probe
Constant pressure probe
 Limitation: data readout and storage is inaccurate.

14.  3.Third generation probe:(Automated probes)
Computer assisted direct data capture was an important step in
reducing examiner bias and also allowed for generation of probe
precision. (according to NIDCR criteria)
e.g.:
 Toronto automated probe
 Florida probe,
 Interprobe,
 Foster Miller probe.

15. FP Handpiece tip as it enters the sulcus
Handpiece tip with constant force in
use (tip at bottom of sulcus) and
sleeve properly positioned at the
top of the gingival margin allowing
the computer to measure the
difference.

17.  4.Fourth generation probes: (Three dimensional probes)
• Currently under development, these are aimed at recording
sequential probe positions along a gingival sulcus.
• An attempt to extend linear probing in a serial manner to take
account of the continuous and three dimensional pocket that is
being examined.
 5.Fifth generation probe: (3D + Noninvasive)
• Basically these will add an ultrasound to a fourth generation
probes.
• If the fourth generation can be made, it will aim in addition to
identify the attachment level without penetrating it.
• e.g.: Ultra sonographic probe.

18. Advances in
RadiographicAssessment

19.  Dental Radiographs are traditional method to assess destruction of
alveolar bone.
“Conventional radiographs are very specific but lack sensitivity”
 Primary criterion for bone loss is the distance from CEJ to the alveolar
crest and distance more than 2 mm is considered as the bone loss.
 But variability affecting conventional radiographic technique are,
 Variation in projection geometry
 Variation in contrast and density
 Masking by other anatomic structures.

20. 1. Digital radiography
 Capturing radiographic image using a sensor
 The first direct digital imaging system, RadioVisioGraphy (RVG), was
invented by Dr. Frances Mouyens.
Advantages
1. Elimination of chemical processing
2. Increased efficiency and speed of viewing
3. Diagnostic information can be enhanced
4. Computerized storage of radiographs
5. Reduced exposure to the radiation

21. 2. Subtraction radiography
 Subtraction radiography was introduced to dentistry in 1980 by
Ruttimann, Webber et & Grondahl HG
 This is a technique by which images not of diagnostic value in a
radiograph, are eliminated so that changes in the radiograph can be
precisely detected
Serial
radiographs
converted to
digital images
superimposed
composite
image
Quantitative
changes

22.  This technique requires a paralleling technique to obtain a standardize
geometry and accurate superimposable radiographs
 This technique facilitates both quantitative and qualitative visualization of
even minor density changes in the bone
 Bone gain appears as light areas and bone loss appears as dark areas
 Rethman et al.(1985): increased detectability of small osseous lesions by
substraction method compared with conventional radiography

23.  Recent image subtraction:“diagnostic subtraction radiography” (DSR)
Modification
 Use of a positioning device during film exposure
 Image analysis software system applies an algorithm to correct angular
alignment discrepancies.

24. 3. Computer Assisted Densitometric Image Analysis (CADIA)
Advantage:
 Measures quantitative changes in
bone density longitudinally.
 Higher sensitivity, reproducibility
and accuracy as compared to
DSR.

26. 4. Computed tomography (CT)
 In 1972, Godfrey Hounsfield announced the invention of a
revolutionary imaging technique, which he referred to as
“computerized axial transverse scanning”
 Fan shaped X-ray source is used
 The computed tomographic image is reconstructed by computer, which
mathematically manipulates data obtained from multiple projections.
 Computed tomography is a specialized radiographic technique that
allows visualization of planes or slices of interest such as axial,
coronal, or sagittal planes referred to as multiplanar imaging.

27. Coronal
CT
Sagittal
CT
Axial CT
Multiplaner imaging

28. Cone-beam Computed Tomography
 Routine use of CT in dentistry is not accepted due to its cost, excessive
radiation, and general practicality.
 In recent years, a new technology of cone-beam CT (CBCT) for
acquiring 3D images of oral structures is now available to the dental
clinics and hospitals.
 It is cheaper than CT, less bulky and generates low dosages of X-
radiations.
 The innovative CBCT machine designed for head and neck imaging are
comparable in size with an orthopantomogram.

30. Advantages
 It gives complete 3D reconstruction
 CBCT units reconstruct the projection data to provide
interrelational images in three orthogonal planes (axial, sagittal,
and coronal).
 Its beam collimation enables limitation of X-radiation to the area
of interest.
 Patient radiation dose is five times lower than normal CT, as the
exposure time is approximately 18 seconds, that is, one-seventh
the amount compared with the conventional medical CT.
 Reduced image artefacts

31. Advances In
MicrobiologicAnalysis

32. Advances In Microbiologic Analysis includes:
1. Immunohistodiagnostic methods
2. Enzymatic methods
3. Molecular biology techniques
 Neucleic acid probes
 Checkerboard DNA-DNA hybridization
 PCR

33. Sample collection
 It is a common need of all the microbiologic analysis to
collect an appropriate subgingival plaque sample
 Mombelli et al. (2002) have shown that four individual
subgingival specimens, each from the deepest
periodontal pocket in each quadrant, should be pooled to
be able to detect the highest amount of pathogens.
 Transport the specimen in a anaerobic environment

34. “Immunodiagnostic methods”
 Immunological assays use fluorescent conjugated antibodies that
recognize specific bacterial antigens, and the identification of
these specific antigen-antibody reactions allows the detection of
target microorganisms.
 This reaction can be visualized using a variety of techniques and
reactions:
1. Direct (DFA) and indirect (IFA) immunofluorescent assays
2. Flow cytometry
3. Enzyme-linked immunosorbent assay (ELISA)
4. Latex agglutination

35. 1. Immunofluorescent assays

36.  IFA is used mainly to detect A.a and P.g
 Zambon et. al (1986) showed that IFA is comparable to
bacterial culture in its ability to identify these pathogens
 Zambon et. al (1995) sensitivity of these assays ranges from
82%-100% for A.a. and 91%-100% for P.g
 Specificity values of 88%-92% and 87%-89% respectively

37. 2. Flow cytometry
 Rapid identification
 Principle is labelling bacterial cells with both species-
specific
antibody and a second fluorescein-conjugated antibody
 This suspension is introduced into flowcytometer, which
separates bacterial cells into an almost single cell
suspension
 Limitation is sophistication and cost involved with
this procedure

38. 3. ELISA

39.  ELISA has been used primarily to detect serum antibodies
to periodontal pathogens.
 In research studies to quantify specific pathogens in
subgingival samples
 A novel chair side ELISA commercially known as
“Evalusite” has been marketed in Europe and Canada for
the chair side detection of 3 periodontal pathogens. Aa,
Pg and Pi

40. 4. Latex agglutination
Test
+ve

41. “Enzymatic Methods”
Tf, Pg, Td, and Capnocytophaga species share common enzymatic
profile- a trypsin like enzyme.
N-benzoyl-d L-arginine-
2-naphthylamide
Trypsin
like
enzyme
BANA hydrolysis
β-naphthylamide
(chromophore)

42.  PERIOSCAN uses this reaction for the identification of this bacterial
profile in plaque isolates
 Loesh et al. (1986) detection of these pariodontal pathogens by BANA
reaction serves as a marker of disease activity
 He also showed that shallow pockets exhibited 10% positive BANA
reaction, whereas deep pockets (7mm) exhibited 80%-90% +ve BANA
reaction
 Beck et al. (1995) used BANA test as a risk indicator for periodontal
attachment loss

43. Disadvantage of BANA
 May be positive in clinically healthy site
 Can not detect sites undergoing periodontal destruction
 Limited organisms detected
 So that, negative results does not rule out the presence of
other important periodontal pathogens.

44. “Molecular Biology Techniques”
 The principles of molecular biology technique reside in the
analysis of DNA, RNA and the structure and function of proteins
 Diagnostic assays require specific DNA fragment that recognize
complementary-specific DNA sequences from target
microorganisms
 This technology requires bacterial DNA extracted from the plaque
sample and amplification of the specific DNA sequence of the
target pathogen

45. 1. Nucleic acid probes
 A probe is a known, single
stranded
(DNA or
nucleic acid molecule
RNA) from a specific
pathogen synthesized and labeled
with a enzyme of a radio isotope
 Hybridization: Pairing of
complimentary strands of DNA to
produce a double stranded DNA.
Pobe
DNA
B.DNA
Hybridization

46.  DMDx and Omnigene are commercially available genomic probes
for the detection of Aa, Pg, Pi and Td.
 Van Steenberghe et al. (1999) reported a sensitivity of 96% and
specificity of 86% for Aa., and 60% and 82% respectively for Pg in
pure lab isolates.
 In clinical specimens, both sensitivity and specificity were reduced
significantly, suggestive of cross reactivity with non target bacteria
in plaque sample because of the presence of homologues
sequences between different bacterial species

47. 2. Checkerboard DNA-DNA hybridization
technology
 Developed by Socransky et.al in 1994
 40 bacterial species can be detected using
whole genomic digoxigenin-labeled DNA
probes.
 Applicable for epidemiologic research and
ecological studies

48. 3. Polymerase chain reaction (PCR)
 Repeated cycles of oligonucleotide (primer)–directed
DNA synthesis of “target sequences” are carried out in
vitro.
 The PCR method is considered the fastest and most
sensitive method available for detecting the presence of
bacterial DNA sequences
 A modification of the original PCR technology, "real-
time" PCR, permits not only detection of specific
microorganisms in plaque, but also its quantification.

49. Advantages
1. High detection limit. As less as 5- 10 cells can be amplified and
detected.
2. Less cross reactivity under optimal conditions
3. Many species can be detected simultaneously
Disadvantage
1. Small quantity needed for reaction may not contain the necessary
target DNA
2. Plaque may contain enzymes which may inhibit these reactions.

50. Advancesin
characterizingthe
Hostresponse

51.  Assessment of host response refers to the study of mediators by
immunologic or biochemical methods, that are recognized as a part of
individual’s response to the periodontal infection.
Mediators
1. specific Mediator
 antibody to a putative pathogen
2. less specific reaction
 the local release of the inflammatory mediators, host derived
enzymes and tissue breakdown products

52. For that...
 Diagnostic tests have been developed that add measures of the
inflammatory process to conventional clinical measures.
 Sources of the sample are:
 GCF, gingival crevicular cells, Saliva, Blood serum, blood cells and
rarely urine.
 Most efforts to date have been based on use of components of GCF
and to a lesser extent, saliva and blood

53. Assessment of Host response
Inflammatory mediators and products
Host derived enzymes
Tissue breakdown products

54. 1. Inflammatory mediators and products
 Cytokines present in GCF and investigated as potential
diagnostic markers are:
 TNF α
 IL-1 α
 IL-1β
 IL-6
 IL-8
 PGE2 (product of COX pathway)
 Cross sectional studies have shown Good correlation with
disease status and severity but not disease progression
 In cases of untreated periodontitis concentration of PGE2
was found increased (showing active Periodontal destruction)

55. Host derived enzymes
 Breakdown of collagen occours by two different pathways:
 Intracellular
1. Aspartate amino transferases
2. Alkaline phosphatase
3. β- Glucuronidase
4. Elastase
 Extracellular
 Matrix metalloproteinase's family (MMPs)

56. Tissue Breakdown Products
 Analysis of GCF obtained from sites with active
periodontitis clearly shows elevated levels of
Hydroxyproline from collagen breakdown and GAGs from
matrix degradation
 Osteocalcin and type-1 collagen peptides- progression of
alveolar bone loss

57. Conclusion

58. 1. This discussion directly translates into improved periodontal therapy
by offering the clinician, the radiographic & laboratory measure of
periodontal infection as an adjunct to traditional clinical indices of
periodontal disease.
2. There is still a lack of a
proven “gold standard” of disease progression
3. But, despite excellent progress in diagnostic
methodology,conventional efforts evaluating inflammation and past
evidence of tissue breakdown remain the standard for disease
evaluation

59. diagnostic test that has demonstrated high predictive value for
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