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GOOD MORNING 1Dr Ashish U. Bisane
Advanced Diagnostic
Aids
2By: Dr Ashish U. Bisane
Introduction
• Periodontal disease is considered a site
specific disease characterized by local
inflammatory reaction to bacterial infection.
• The attempts to identify specific bacteria,
specific inflammatory cells or molecules, or
specific breakdown products have been the
soul reason for the advances in diagnostic
methodology.
3Dr Ashish U. Bisane
Gingival Bleeding
• Gingival Inflammation is diagnosed based upon:
1. Gingival Bleeding on Probing (Objective sign)
2. Gingival Redness
3. Gingival edema/swelling
• 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. (Greenstein G, 1981; Polson AM, 1985)
5Dr Ashish U. Bisane
• To test for bleeding
after probing, the probe
is carefully introduced
to the bottom of the
pocket and gently
moved laterally along
the pocket wall.
• Photomicrograph of
thin, microulcerated
epithelial lining of the
soft tissue wall of an
infected pocket. Such
sites readily bleed when
gently probed
6Dr Ashish U. Bisane
• Subjects who refrain from normal oral hygiene procedures have a resultant increase
in plaque accumulation and demonstrate a concomitant increase in gingival
bleeding as gingivitis develops over a 2-3 week period. (Greenstein G, 1981)
• Lang et al 1991, in a retrospective study reported that sites that bled on probing at
several visits had a greater probability of losing attachment than those that bled at
one visit or did not bleed.
• However, well controlled longitudinal studies investigated the predictive values of
such clinical sign, 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. (Badersten.A, 1985; Haffajee.A.D,
1983)
7Dr Ashish U. Bisane
• Drawbacks:
1. Healthy sites may bleed on probing as any force
greater than 0.25N evokes bleeding even in an intact
periodontium.
2. Relationship to disease progress is unclear.
3. Signs of inflammation are inhibited in smokers
(Nair.P, 2003).
8Dr Ashish U. Bisane
(Sub)Gingival Temperature
• Kung et al, 1990; demonstrated that the
subgingival temperature at diseased sites is
increased compared with healthy sites and that a
natural anteroposterior temperature gradient exists
within the dental arches.
• Kung et al, 1990; thus, claimed that thermal
probes are sensitive diagnostic devices for
measuring early inflammatory changes in the
gingival tissues.
9Dr Ashish U. Bisane
Perio-Temp (Thermal
Periodontal Probing System)
• Perio-Temp Probe (Abiodent)
• Enables the calculation of temperature
differential with a sensitivity of 0.10celcius.
• Individual temperature differences are
compared with those expected for each tooth,
and higher temperature pockets are signaled
with a red-emitting diode.
10Dr Ashish U. Bisane
Perio-Temp
11Dr Ashish U. Bisane
• 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.
• Haffajee et al, 1992; also found that elevated subgingival site temperature was
particularly related to attachment loss in shallow pockets and that P. intermedia, P.
micros, P. gingivalis, T. forsythia and Aac had elevated proportions in the total
microbiota in sites with elevated temperatures.
• Trikilis.N, 1999; reported differences in the temperature differentials between
subgingival temperature and sublingual temperature in smokers compared to non-
smokers.
12Dr Ashish U. Bisane
• The reason why temperature increases with probing
depth is unclear. A possible explanation is an increase
in cellular and molecular activity caused by increased
periodontal inflammation with increased probing depth.
• Whether the pathogens are responsible for higher
temperature by initiating the inflammatory process, or
whether the increased temperature provides an
environment favourable for the pathogens, remains
unclear.
13Dr Ashish U. Bisane
Periodontal Probing
• Currently the “Gold Standard” for recording changes in
periodontal status in longitudinal measurement of
clinical attachment levels.
• Factors affecting probing measurements: (Listgarten.
M. A, 1976)
1. Probing technique.
2. Probing force.
3. Size of the probe tip.
4. Angle of insertion of the probe.
5. Precision of probe calibration.
14Dr Ashish U. Bisane
Drawbacks in manual
probing:
1. Use of the periodontal probe - many problems in terms of sensitivity and
reproducibility of the measurements.
2. Clinical pocket depth obtained does not normally coincide with the histologic
pocket depth.
3. Inflammed tissue offers less resistance to probe penetration, and the probe tip
either coincides with or is apical to the coronal level of connective tissue
attachment. (Listgarten. M. A, 1976)
4. Healed gingiva and post subgingival instrumentation, there is increased resistance
to periodontal probing. (Magnusson. I, 1980)
15Dr Ashish U. Bisane
Probing Force
• Penetration of probe has been positively
correlated with probing force. (Mombelli.A,
1986)
• With forces up to 30g, the tip of probe seems
to remain within the intact junctional
epithelium (Armitage. G.C, 1994)
• Forces up to 50g are necessary to diagnose
periodontal osseous defects (Kalkwarf.K.L,
1986)
16Dr Ashish U. Bisane
Classification
• First generation probes/ conventional
• Second generation probes/pressure sensitive
• Third generation probes/ computerized
Standardization of probe tip (<1mm) and use
of registration stents have been suggested to
maintain reproducible angulations and reduce
the error. (Badersten.A. 1984)
17Dr Ashish U. Bisane
18Dr Ashish U. Bisane
National Institute of Dental and Craniofacial Research (NIDCR) criteria
for overcoming limitations of conventional periodontal probing (Parakkal. P.F,
1979)
Limitation Conventional Probing NIDCR Criteria
1. Precision 1mm 0.1 mm
2. Range 12 mm 10 mm
3. Probing Force Non standardized Constant & Standardized
4. Applicability Non-invasive and easy to use Non-invasive , light weight
and easy to use
5. Reach Easy to access any location around all teeth Easy to access any location
around all teeth
6. Angulation Subjective A guidance system to ensure
proper angulation.
7. Security Easily sterilized.
Simple stainless steel instruments..
Complete sterilization of all
the portions entering the
mouth.
No biohazard from material or
electric shock.
8. Readout Depending upon voice dictation and
recording in writing
Direct electronic reading and
digital output. 19Dr Ashish U. Bisane
Florida Probe System
• Gibbs et al - developed Florida Probe System (Florida Probe
Corporation)
• Parts of Florida Probe System:
1. Probe handpiece
2. Digital readout
3. Foot switch
4. Computer interface
5. Computer
• 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.
20Dr Ashish U. Bisane
Advantages
1. Constant probing force
2. Precise electronic measurement
3. Computer storage of data
Disadvantages
1. Lack of tactile sensitivity - independent movement, which forces
the operator to predetermine an insertion point and angle.
2. Use of a fixed force setting throughout the mouth, regardless of the
site or inflammatory status, may generate inaccurate measurements
or patient discomfort.
3. 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. (Perry. D.A. 1994)
21Dr Ashish U. Bisane
22Dr Ashish U. Bisane
23Dr Ashish U. Bisane
Florida Probe System to
Record CAL
• CALs are recorded relative to a fixed reference
point, such as the occlusal surfaces of the teeth
(Disk Probe) or a Prefabricated stent (Stent
Probe).
• These measurements are made sequentially
over time, and thus differences in relative
attachment levels at consecutive examinations
must be calculated.
24Dr Ashish U. Bisane
25Dr Ashish U. Bisane
Florida PASHA Probe
• Modification of Florida probe to detect CEJ.
• Has a Modified Sleeve, which includes a
prominent 0.125 mm edge to facilitate a “catch”
of the CEJ.
• The width of this edge is considered small enough
not to interfere with probing depth measurement,
offering clinicians to measure CAL and probing
depth concurrently.
• Reliably reproduces and identifies CEJ in human
skulls (Preshaw.P.M, 1999) and shows promise in
measuring CALs in humans (Karpinia. K, 2004)
26Dr Ashish U. Bisane
Toronto Automated Probe
• The uses the occlusal-incisal surface to measure
relative clinical attachment levels.
(Mcculloch.C.A.1991)
• The sulcus is probed with a 0.5mm nickel-
titanium wire that is extended under air pressure.
• It controls angular discrepancies by means of a
mercury-tilt sensor that limits angulation within +
30 degrees, but it requires reproducible
positioning of patient’s head and cannot easily
measure 2nd and 3rd molars.
27Dr Ashish U. Bisane
Interprobe & Perioprobe
• 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
28Dr Ashish U. Bisane
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.
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30Dr Ashish U. Bisane
31Dr Ashish U. Bisane
Drawbacks of Conventional
Radiography
• Conventional radiographs are very specific but lack
sensitivity.
• Considerable amount of bone loss should occur for being
visible on the radiographs.
• Causes of variability in conventional radiographic
techniques:
1. Variations in projection geometry.
2. Variations in contrast and density caused by differences in
film processing, voltage and exposure time.
3. Masking of osseous changes by other anatomic structures.
32Dr Ashish U. Bisane
Solutions for Standardization
of Radiographic Techniques
• Radiographs should be obtained in a constant
reproducible plane using film holders with a
template containing some type of template
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 (Rosling. B. 1975)
33Dr Ashish U. Bisane
Digital 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 ½ of dose reduction compared with conventional
radiographs).
• Telediagnosis, video conferencing, rapid image
transmission are some of the other advantages.
34Dr Ashish U. Bisane
Types of Digital Intraoral Radiographic
Systems (Parks.E.T. 2002)
• First system
1. Charge – Coupled Devices (CCDs)
2. Complementary Metal Oxide Semiconductor (CMOS)
receptors as detectors.
• These devices are placed in the patient’s mouth and are
linked by a wire to the computer.
• On radiation exposure the radiographic image appears
on computer screen.
• The detector is then moved to the next position and so
on until the complete area to be imaged is covered.
35Dr Ashish U. Bisane
• Second System: uses Photostimulable Phosphor (PSP) plates as
detectors.
• PSP plates resemble film with one of the sides lined with a PSP
coating.
• When interacting with x-rays, PSP stores energy, which it then
releases on stimulation by light of an appropriate wavelength.
• PSP plates are placed and exposed similar to regular film.
• The exposed plates are placed on a plate scanner and scanned by a
laser beam, and the radiographic image appears on the computer
screen.
36Dr Ashish U. Bisane
The Digital Advantage
1. Speed of image capture and display
2. Low x-ray exposure
3. Ability to manipulate the image and maximize
diagnostic efficacy.
4. Use of digital tools such as linear, angular and
density measurements.
5. Improved patient education
6. Ease of storage, transfer and copying.
7. Seamless integration with electronic patient
record management or other software
38Dr Ashish U. Bisane
• Once captured and displayed, computer software can be
used to enhance the digital image and increase its diagnostic
efficacy. (Preston J D, 1999)
• Exposure can be adjusted.
• A sharpening (Edge Enhancement) filter increases the
definition and separation of adjacent structures.
• Inversion filters provide a negative of the image that might
sometime reveal diseases not seen on the positive images.
• Areas of interest can be magnified.
• Different image qualities allow better detection of dental
diseases. (High contrast for caries and Low contrast for
Alveolar Crest detection)
39Dr Ashish U. Bisane
Subtraction Radiography
• This technique requires a paralleling technique to obtain a
standardized geometry and accurate superimposable radiographs.
• It relies on the conversion of serial radiographs into digital images.
• The serially obtained digital images can be superimposed and the
resultant composite can be viewed on a video screen.
• Changes in the density and volume of bone can be detected as
1. Lighter areas (Bone Gain)
2. Dark Areas (Bone Loss)
• It facilitates both quantitative and qualitative visualization of even
minor density changes in bone by removing the unchanged
anatomic structures from the image.
40Dr Ashish U. Bisane
Advantages:
High correlation between alveolar bone loss
and CAL changes
Increased detection of small osseous lesion
Both quantitative and qualitative visualization
More sensitive
41Dr Ashish U. Bisane
• Studies using this technique have shown:
1. A higher degree of correlation between changes
in alveolar bone determined by subtraction
radiography & CAL changes in periodontal
patients after therapy. (Hausmann E, 1985)
2. Increased detectability of small osseous lesions
compared with conventional radiographs from
which the subtraction images are produced.
(Rethman M, 1985)
42Dr Ashish U. Bisane
Serial radiographs
converted to digital images
superimposed
composite image
Quantitative changes 43Dr Ashish U. Bisane
• The disadvantage is the need to be almost at
identical projection alignment during the
exposure of sequential radiographs which
makes this method impractical in clinical
setting.
44Dr Ashish U. Bisane
Diagnostic/Digital
Subtraction Radiography (DSR)
• Combines the use of a positioning device during
film exposure with a specialized software
designed for digital image subtraction using
conventional personal computers in dental offices.
• The software corrects the effects of angular
alignment discrepancies and provides some
degree of flexibility in the imaging procedure.
• DSR showed statistically significant gains in
diagnostic accuracy over conventional
radiographs but no differences with Subtraction
radiography. (Nummikoski PV, 2000)
45Dr Ashish U. Bisane
Computer Assisted Densitometric
Image Analysis System (CADIA)
• A video camera measures the light transmitted through
the radiograph and signals from the camera are
converted into gray scale images (Bragger U, 1988)
• The camera is interfaced with an image processor and a
computer that allows the storage and mathematic
manipulation of the images.
• CADIA offers an objective method for following
alveolar bone density changes quantitatively over time.
• It has also shown higher sensitivity and a higher degree
of reproducibility and accuracy than DSR.
46Dr Ashish U. Bisane
Advances in
Microbiological
Analysis
47Dr Ashish U. Bisane
• Despite difficulty in identifying all the members of the oral microbiota and
understanding how they interact with each other and with the host, a
limited number of micro-organisms have demonstrated a clear etiologic
role and have been identified as periodontal pathogens. (Genco RJ, 1996)
• Microbiologic tests to identify the putative organisms have the potential
to support the diagnosis of 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.
• Microbiologic tests can also be used to monitor periodontal therapy
directed at the suppression or eradication of periodontal pathogenic
micro-organisms.(Van Winkelhoff AJ, 1996)
48Dr Ashish U. Bisane
Bacterial Culturing
• Gold Standard
• Plaque samples are cultivated under anaerobic conditions using
selective and non-selective media.
• Advantage:
1. Relative and Absolute count of the cultured species.
• Disadvantage:
1. Strict sampling conditions
2. Difficulty in culturing most organisms
3. Low sensitivity : organisms lesser then 103 is difficult to detect
4. Time consuming
5. Expensive equipment and experienced personnel
49Dr Ashish U. Bisane
Direct Microscopy
• Dark field or phase contrast microscopy.
• Most of the periodontal pathogens are non-
motile so it is difficult to identify.
• Even different species of Treponema cannot be
distinguished by direct microscopic
techniques.
50Dr Ashish U. Bisane
Immunodiagnostic Methods
• Immunologic assays employ antibodies that
recognize specific bacterial antigens to detect
target micro-organisms.
1. Direct and Indirect Immunofluoroscent
(microscopic) Assays
2. Flow Cytometry
3. Enzyme Linked Immunosorbent Assay
(ELISA)
4. Latex Agglutination
51Dr Ashish U. Bisane
Direct Immunofluoroscent
Assay (DFA)
AB conjugated
with Fluorescein
marker
Bacteria (Antigen)
Immunofluoroscent
complex detectable
under microscope
52Dr Ashish U. Bisane
Indirect Immunofluoroscent
Assay
Primary
AB
Bacterial
AG
Immune
Complex
Secondary
Fluoroscein
conjugated
Antibody
Stains the
primary
AG:AB
complex
53Dr Ashish U. Bisane
Direct IFA
Indirect IFA
54Dr Ashish U. Bisane
Flow Cytometry or
Cytofluorography
Bacterial cells + species specific antibody
+ Secondary Fluorescein Conjugated
antibody
Introduced in flowcytometer.
Bacterial cells are separated into single
cell suspension by means of laminar flow.
Cells identified by lasers.
55Dr Ashish U. Bisane
ELISA
• Similar to Antibody and Antigen reaction, but the
fluorescence, instead of a radioisotope, is read using an
enzymatically driven color reaction with a photometer.
• Intensity of color depends upon the concentration of the
antigen and is usually read photometrically for optimal
quantification.
 Evalusite: commercially available chairside diagnostic
kit to detect Aa, Pg and Pi.
 It involves linkage between antigen and a membrane
bound antibody to form an immunocomplex that is
revealed later with a colorimetric reaction.
56Dr Ashish U. Bisane
57Dr Ashish U. Bisane
58Dr Ashish U. Bisane
Latex Agglutination
• Based on binding of protein to latex.
Latex beads coated with species specific antibody
When beads come in contact with specific species in sample they bind and agglutination
occurs
Clumping of beads is visible
Test positive 59Dr Ashish U. Bisane
Enzymatic Methods
• Bacteria release specific enzymes.
• Certain group of species share common
enzymatic profile.
• E.g. Tf, PG, Td, and Capnocytophaga species
release trypsin-like enzyme.
• The activity of this enzyme is measured with
the hydrolysis of the colorless substrate:
 N-benzoyl-d L-arginine-2-naphthylamide
(BANA)
60Dr Ashish U. Bisane
BANA Test
BANA Substrate
Hydrolysis by
Trypsin like
enzyme of bacteria
Hydrolysis
Formation of
Chromophore: B-
naphthylamide
Addition of Fast
Garnet
The chromophore
turns orange red
61Dr Ashish U. Bisane
Perioscan is a popular diagnostic kit uses
BANA reaction.
Disadvantages:
May be positive in clinically healthy site
Cannot detect disease activity
Limited organisms detected
Other pathogens may be present if it’s
negative.
62Dr Ashish U. Bisane
Molecular Biology for
Diagnosis
• Based on analysis of
DNA, RNA and
structure & function
of Protein of the
micro-organisms.
• A “probe” is a known
nucleic acid molecule
(DNA or RNA) from
a specific micro-
organism artificially
synthesized and
labeled for its
detection when placed
with a plaque sample.
63Dr Ashish U. Bisane
Nucleic Acid Probes
• A DNA probe uses a segment of a single
stranded DNA, labeled with a enzyme of a
radio isotope, that is able to hybridize to a
complimentary nuclei strand, and thus detect
presence of target microorganism.
• Two types:
1. Whole genomic probes
2. Oligonucleotide probes
64Dr Ashish U. Bisane
Whole Genomic Probes
• Targets the whole DNA strand rather then a
specific sequence or gene.
• High chances to cross react with non target
microorganism
• Lower sensitivity and specificity.
• Whole genomic probes for Aa, Pg, Pi and Td
have been commercially developed and tested
E.g.DMDx, Omnigene.
65Dr Ashish U. Bisane
66Dr Ashish U. Bisane
Oligonucleotide Probes
• Target variable region of 16sRNA or a specific
sequence in the DNA strand.
• Higher sensitivity and specificity.
• These bacterial 16s RNA genes contain both
regions shared by different bacteria and short
stretches of variable regions shared by only
specific micro-organisms of the same species
or genus. (Moncla BJ, 1990)
67Dr Ashish U. Bisane
Hybridization
• Pairing of complementary strands of DNA
(nucleic acid) to produce a double stranded
DNA (nucleic acid).
• All hybridization methods use radiolabeled or
fluoroscence-labeled DNA probes that bind to
the target DNA of interest, thus allowing its
visualization.
68Dr Ashish U. Bisane
Checkerboard DNA-DNA
Hybridization Technology
• Developed by Socransky et.al. 1994.
• 40 bacterial species can be detected using
whole genomic digoxigenin-labeled DNA
probes.
• Large number of samples can be tested and up
to 40 oral species detected with a single test.
• Highly specific test.
• Requires expensive sophisticated laboratory
equipment.
69Dr Ashish U. Bisane
• Advantages of DNA probes as compared to
bacterial culturing.
1. More sensitive and specific
2. Requires as less as 104 cells of each species to
be detected.
3. Multiple species detected with a single test
4. Does not require viable bacteria
5. Large number of samples can be assessed.
• Disadvantages:
1. Expensive
2. Expert personnel to carry out the test
3. Not easily available
70Dr Ashish U. Bisane
Polymerase Chain Reaction
• Developed in 1985.
• Used almost universally to study DNA or RNA
obtained from a variety of tissue sources.
• Involves amplification of a region of DNA by
a primer specific to the target species.
• If there is amplification then it indicates the
presence of the target species in the sample.
71Dr Ashish U. Bisane
72Dr Ashish U. Bisane
 Isolation of DNA from a fresh tissue specimen.
 Heating of complementary double strands of DNA.
 DNA splits into single stranded forms.
 Single stranded forms are intended to act as template dictating the nucleotide sequencing in vitro.
 Amplification is done using a DNA polymerase that requires a primer or a known short
oligonucleotide sequence complementary to the border of the region that is to be amplified.
 To obtain amplified fragments of constant length and in large quantities, a second primer,
complementary of the opposed chain must be used to bind the template and flank the region of
interest.
 This amplification can be performed several number of times known as cycles.
 This sequenced DNA is then detected and visualized through electrophoresis in aragose gel and
ethidium-bromure obtaining a qualitative signal.
73Dr Ashish U. Bisane
• In 1988, a thermo-stable DNA polymerase
isolated from the organism Thermus aquaticus
, known as Taq-polymerase was developed.
• This Taq-polymerase has allowed
automatization of the reaction using specific
appliances called thermocycles.
74Dr Ashish U. Bisane
75Dr Ashish U. Bisane
Real Time PCRs
• To quantitatively assess target bacteria using
PCR technique.
• By using a single copy of the bacterial genes
per cell, a good correlation between the
fluorescent signal measured and the number of
cells can be obtained. (Lyons SR,2000; Smith
AJ, 1996)
76Dr Ashish U. Bisane
• Advantages:
1. High degree of sensitivity and specificity
2. Reproducible and consistent
3. High detection limit. As less as 5- 10 cells can be amplified and
detected.
4. Less cross reactivity under optimal conditions.
5. Many species can be detected simultaneously.
• Disadvantage:
1. Expensive lab equipments
2. Small quantity needed for reaction may not contain the necessary
target DNA.
3. Plaque may contain enzymes which may inhibit these reactions.
77Dr Ashish U. Bisane
Advances in Characterizing
Host Response
• Asses host response by studying mediators as a
response to specific bacteria or local release of
inflammatory mediators or enzymes as response to
infection.
• Source of samples may be; GCF, Saliva, or Blood.
• GCF is most commonly used, where as saliva is been
recently been researched recently.
1. Host derived enzymes
2. Inflammatory mediators and products
3. Tissue Breakdown Products
78Dr Ashish U. Bisane
GCF
• Most well studied, with almost 40 components in form
of host-derived enzymes, tissue breakdown products,
and inflammatory mediators.
• Collected with paper strips, micro papillary tubes,
micropipettes, microsyringes, plastic strips.
• Paper strips commonly used, introduced in sulcus for
30 secs and volume is measured using Periotron 6000.
• Periotron measures the capacitance across the wet
paper strip, which is converted to digital reading.
• Periotron reading have high correlation with clinical
gingival indices.
• Quickest and easiest way to measure GCF.
79Dr Ashish U. Bisane
Perio Paper Strips
Periotron 8000
80Dr Ashish U. Bisane
Saliva
• The next most used after GCF.
• Easily collected
• Contains both local and systemic derived markers for periodontal diseases.
• Collected from parotid, sub-mandibular or sub lingual or as ‘Whole saliva’.
• Whole saliva contains secretions of major and minor salivary glands,
desquamated cells, and GCF.
• Markers to look for in saliva:
1. Proteins and enzymes from host,
2. Phenotypic markers,
3. Host cells,
4. Hormones,
5. Bacteria, bacterial products,
6. Volatile compounds, and ions.
81Dr Ashish U. Bisane
Cytokines
• Are substances released by cells of the
immune system.
• Cytokines in GCF are: TNF-alpha, IL-1, IL-6,
and IL-8
• Can be used to determine the disease activity.
• Esp. Prostaglandin E in increased in GCF of
periodontitis patients.
• Can be used to determine disease activity
82Dr Ashish U. Bisane
Host derived enzymes
1. Aspartate aminotrasnferase( AST),
2. Alkaline phosphatase,
3. B-glucoronidase,
4. Elastase,
5. Cathespins,
6. MMPs.
83Dr Ashish U. Bisane
AST
• Derived from dead cells,.
• Elevated in periodontal disease
• Periogard is a commercially available colorimetric test
for AST.
• It involves GCF collection with a filter paper strip, which is
then placed in a tromethamine hydrochloride buffer. A
substrate reaction mixture containing L-aspartic and a-
ketoglutaric acids are added and allowed to react for 10min.
In presence of AST, aspartate and glutarate are catalyzed to
oxaloacetate and glutamate. Addition of a dye (Fast Red)
results in a colour product. The intensity of which is
proportional to the AST activity in GCF sample.
• However, AST levels cannot differentiate active and
inactive sites.
84Dr Ashish U. Bisane
• ALP: released from osteoblast, neutrophils,
fibroblast..
• Beta Glucoronidase and Elastase: found in
Neutrophils.
• Beta Glucoronidase, Elastase, Neutral protease,
and Cathepsins:
All are shown to be higher in diseased sites. May
be used to predict severity of disease or to predict
disease activity.
85Dr Ashish U. Bisane
Matrix metalloproteinases
• zinc and calcium dependent enzymes
• constitutively formed in the body, secreted by fibroblast
and macrophages.
• Normally help in degrading and remodeling of
extracellular matrices.
• In chronic periodontitis they cause the degradation of
the collagen fibrils in PDL and Alveolar bone.
• MMP,2,3,8 9 and 13 play important role.
• MMP8 level is associated with the attachment loss
• Level reduces in response to treatment. (Chair side test
kit)
• Can be used to indicate present disease status and
predictor of future disease.
86Dr Ashish U. Bisane
Tissue Breakdown Products
• Analysis of GCF obtained from sites with
periodontitis clearly show elevated levels of
hydroxyproline from collagen breakdown and
GAGs from matrix degradation. (Last KS,
1985)
• Osteocalcin and Type-I collagen peptides.
87Dr Ashish U. Bisane
Conclusion
• No proven marker available to predict the
disease activity as there is no proven
correlation between these markers and the
clinical loss of attachment.
• Search of tool with high predictive value,
simple, safe and cost effective still needed.
88Dr Ashish U. Bisane
Thank You !!
89Dr Ashish U. Bisane

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Advanced diagnostic aids 24 9-12

  • 1. GOOD MORNING 1Dr Ashish U. Bisane
  • 3. Introduction • Periodontal disease is considered a site specific disease characterized by local inflammatory reaction to bacterial infection. • The attempts to identify specific bacteria, specific inflammatory cells or molecules, or specific breakdown products have been the soul reason for the advances in diagnostic methodology. 3Dr Ashish U. Bisane
  • 4. Gingival Bleeding • Gingival Inflammation is diagnosed based upon: 1. Gingival Bleeding on Probing (Objective sign) 2. Gingival Redness 3. Gingival edema/swelling • 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. (Greenstein G, 1981; Polson AM, 1985) 5Dr Ashish U. Bisane
  • 5. • To test for bleeding after probing, the probe is carefully introduced to the bottom of the pocket and gently moved laterally along the pocket wall. • Photomicrograph of thin, microulcerated epithelial lining of the soft tissue wall of an infected pocket. Such sites readily bleed when gently probed 6Dr Ashish U. Bisane
  • 6. • Subjects who refrain from normal oral hygiene procedures have a resultant increase in plaque accumulation and demonstrate a concomitant increase in gingival bleeding as gingivitis develops over a 2-3 week period. (Greenstein G, 1981) • Lang et al 1991, in a retrospective study reported that sites that bled on probing at several visits had a greater probability of losing attachment than those that bled at one visit or did not bleed. • However, well controlled longitudinal studies investigated the predictive values of such clinical sign, 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. (Badersten.A, 1985; Haffajee.A.D, 1983) 7Dr Ashish U. Bisane
  • 7. • Drawbacks: 1. Healthy sites may bleed on probing as any force greater than 0.25N evokes bleeding even in an intact periodontium. 2. Relationship to disease progress is unclear. 3. Signs of inflammation are inhibited in smokers (Nair.P, 2003). 8Dr Ashish U. Bisane
  • 8. (Sub)Gingival Temperature • Kung et al, 1990; demonstrated that the subgingival temperature at diseased sites is increased compared with healthy sites and that a natural anteroposterior temperature gradient exists within the dental arches. • Kung et al, 1990; thus, claimed that thermal probes are sensitive diagnostic devices for measuring early inflammatory changes in the gingival tissues. 9Dr Ashish U. Bisane
  • 9. Perio-Temp (Thermal Periodontal Probing System) • Perio-Temp Probe (Abiodent) • Enables the calculation of temperature differential with a sensitivity of 0.10celcius. • Individual temperature differences are compared with those expected for each tooth, and higher temperature pockets are signaled with a red-emitting diode. 10Dr Ashish U. Bisane
  • 11. • 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. • Haffajee et al, 1992; also found that elevated subgingival site temperature was particularly related to attachment loss in shallow pockets and that P. intermedia, P. micros, P. gingivalis, T. forsythia and Aac had elevated proportions in the total microbiota in sites with elevated temperatures. • Trikilis.N, 1999; reported differences in the temperature differentials between subgingival temperature and sublingual temperature in smokers compared to non- smokers. 12Dr Ashish U. Bisane
  • 12. • The reason why temperature increases with probing depth is unclear. A possible explanation is an increase in cellular and molecular activity caused by increased periodontal inflammation with increased probing depth. • Whether the pathogens are responsible for higher temperature by initiating the inflammatory process, or whether the increased temperature provides an environment favourable for the pathogens, remains unclear. 13Dr Ashish U. Bisane
  • 13. Periodontal Probing • Currently the “Gold Standard” for recording changes in periodontal status in longitudinal measurement of clinical attachment levels. • Factors affecting probing measurements: (Listgarten. M. A, 1976) 1. Probing technique. 2. Probing force. 3. Size of the probe tip. 4. Angle of insertion of the probe. 5. Precision of probe calibration. 14Dr Ashish U. Bisane
  • 14. Drawbacks in manual probing: 1. Use of the periodontal probe - many problems in terms of sensitivity and reproducibility of the measurements. 2. Clinical pocket depth obtained does not normally coincide with the histologic pocket depth. 3. Inflammed tissue offers less resistance to probe penetration, and the probe tip either coincides with or is apical to the coronal level of connective tissue attachment. (Listgarten. M. A, 1976) 4. Healed gingiva and post subgingival instrumentation, there is increased resistance to periodontal probing. (Magnusson. I, 1980) 15Dr Ashish U. Bisane
  • 15. Probing Force • Penetration of probe has been positively correlated with probing force. (Mombelli.A, 1986) • With forces up to 30g, the tip of probe seems to remain within the intact junctional epithelium (Armitage. G.C, 1994) • Forces up to 50g are necessary to diagnose periodontal osseous defects (Kalkwarf.K.L, 1986) 16Dr Ashish U. Bisane
  • 16. Classification • First generation probes/ conventional • Second generation probes/pressure sensitive • Third generation probes/ computerized Standardization of probe tip (<1mm) and use of registration stents have been suggested to maintain reproducible angulations and reduce the error. (Badersten.A. 1984) 17Dr Ashish U. Bisane
  • 17. 18Dr Ashish U. Bisane
  • 18. National Institute of Dental and Craniofacial Research (NIDCR) criteria for overcoming limitations of conventional periodontal probing (Parakkal. P.F, 1979) Limitation Conventional Probing NIDCR Criteria 1. Precision 1mm 0.1 mm 2. Range 12 mm 10 mm 3. Probing Force Non standardized Constant & Standardized 4. Applicability Non-invasive and easy to use Non-invasive , light weight and easy to use 5. Reach Easy to access any location around all teeth Easy to access any location around all teeth 6. Angulation Subjective A guidance system to ensure proper angulation. 7. Security Easily sterilized. Simple stainless steel instruments.. Complete sterilization of all the portions entering the mouth. No biohazard from material or electric shock. 8. Readout Depending upon voice dictation and recording in writing Direct electronic reading and digital output. 19Dr Ashish U. Bisane
  • 19. Florida Probe System • Gibbs et al - developed Florida Probe System (Florida Probe Corporation) • Parts of Florida Probe System: 1. Probe handpiece 2. Digital readout 3. Foot switch 4. Computer interface 5. Computer • 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. 20Dr Ashish U. Bisane
  • 20. Advantages 1. Constant probing force 2. Precise electronic measurement 3. Computer storage of data Disadvantages 1. Lack of tactile sensitivity - independent movement, which forces the operator to predetermine an insertion point and angle. 2. Use of a fixed force setting throughout the mouth, regardless of the site or inflammatory status, may generate inaccurate measurements or patient discomfort. 3. 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. (Perry. D.A. 1994) 21Dr Ashish U. Bisane
  • 21. 22Dr Ashish U. Bisane
  • 22. 23Dr Ashish U. Bisane
  • 23. Florida Probe System to Record CAL • CALs are recorded relative to a fixed reference point, such as the occlusal surfaces of the teeth (Disk Probe) or a Prefabricated stent (Stent Probe). • These measurements are made sequentially over time, and thus differences in relative attachment levels at consecutive examinations must be calculated. 24Dr Ashish U. Bisane
  • 24. 25Dr Ashish U. Bisane
  • 25. Florida PASHA Probe • Modification of Florida probe to detect CEJ. • Has a Modified Sleeve, which includes a prominent 0.125 mm edge to facilitate a “catch” of the CEJ. • The width of this edge is considered small enough not to interfere with probing depth measurement, offering clinicians to measure CAL and probing depth concurrently. • Reliably reproduces and identifies CEJ in human skulls (Preshaw.P.M, 1999) and shows promise in measuring CALs in humans (Karpinia. K, 2004) 26Dr Ashish U. Bisane
  • 26. Toronto Automated Probe • The uses the occlusal-incisal surface to measure relative clinical attachment levels. (Mcculloch.C.A.1991) • The sulcus is probed with a 0.5mm nickel- titanium wire that is extended under air pressure. • It controls angular discrepancies by means of a mercury-tilt sensor that limits angulation within + 30 degrees, but it requires reproducible positioning of patient’s head and cannot easily measure 2nd and 3rd molars. 27Dr Ashish U. Bisane
  • 27. Interprobe & Perioprobe • 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 28Dr Ashish U. Bisane
  • 28. 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. 29Dr Ashish U. Bisane
  • 29. 30Dr Ashish U. Bisane
  • 30. 31Dr Ashish U. Bisane
  • 31. Drawbacks of Conventional Radiography • Conventional radiographs are very specific but lack sensitivity. • Considerable amount of bone loss should occur for being visible on the radiographs. • Causes of variability in conventional radiographic techniques: 1. Variations in projection geometry. 2. Variations in contrast and density caused by differences in film processing, voltage and exposure time. 3. Masking of osseous changes by other anatomic structures. 32Dr Ashish U. Bisane
  • 32. Solutions for Standardization of Radiographic Techniques • Radiographs should be obtained in a constant reproducible plane using film holders with a template containing some type of template 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 (Rosling. B. 1975) 33Dr Ashish U. Bisane
  • 33. Digital 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 ½ of dose reduction compared with conventional radiographs). • Telediagnosis, video conferencing, rapid image transmission are some of the other advantages. 34Dr Ashish U. Bisane
  • 34. Types of Digital Intraoral Radiographic Systems (Parks.E.T. 2002) • First system 1. Charge – Coupled Devices (CCDs) 2. Complementary Metal Oxide Semiconductor (CMOS) receptors as detectors. • These devices are placed in the patient’s mouth and are linked by a wire to the computer. • On radiation exposure the radiographic image appears on computer screen. • The detector is then moved to the next position and so on until the complete area to be imaged is covered. 35Dr Ashish U. Bisane
  • 35. • Second System: uses Photostimulable Phosphor (PSP) plates as detectors. • PSP plates resemble film with one of the sides lined with a PSP coating. • When interacting with x-rays, PSP stores energy, which it then releases on stimulation by light of an appropriate wavelength. • PSP plates are placed and exposed similar to regular film. • The exposed plates are placed on a plate scanner and scanned by a laser beam, and the radiographic image appears on the computer screen. 36Dr Ashish U. Bisane
  • 36. The Digital Advantage 1. Speed of image capture and display 2. Low x-ray exposure 3. Ability to manipulate the image and maximize diagnostic efficacy. 4. Use of digital tools such as linear, angular and density measurements. 5. Improved patient education 6. Ease of storage, transfer and copying. 7. Seamless integration with electronic patient record management or other software 38Dr Ashish U. Bisane
  • 37. • Once captured and displayed, computer software can be used to enhance the digital image and increase its diagnostic efficacy. (Preston J D, 1999) • Exposure can be adjusted. • A sharpening (Edge Enhancement) filter increases the definition and separation of adjacent structures. • Inversion filters provide a negative of the image that might sometime reveal diseases not seen on the positive images. • Areas of interest can be magnified. • Different image qualities allow better detection of dental diseases. (High contrast for caries and Low contrast for Alveolar Crest detection) 39Dr Ashish U. Bisane
  • 38. Subtraction Radiography • This technique requires a paralleling technique to obtain a standardized geometry and accurate superimposable radiographs. • It relies on the conversion of serial radiographs into digital images. • The serially obtained digital images can be superimposed and the resultant composite can be viewed on a video screen. • Changes in the density and volume of bone can be detected as 1. Lighter areas (Bone Gain) 2. Dark Areas (Bone Loss) • It facilitates both quantitative and qualitative visualization of even minor density changes in bone by removing the unchanged anatomic structures from the image. 40Dr Ashish U. Bisane
  • 39. Advantages: High correlation between alveolar bone loss and CAL changes Increased detection of small osseous lesion Both quantitative and qualitative visualization More sensitive 41Dr Ashish U. Bisane
  • 40. • Studies using this technique have shown: 1. A higher degree of correlation between changes in alveolar bone determined by subtraction radiography & CAL changes in periodontal patients after therapy. (Hausmann E, 1985) 2. Increased detectability of small osseous lesions compared with conventional radiographs from which the subtraction images are produced. (Rethman M, 1985) 42Dr Ashish U. Bisane
  • 41. Serial radiographs converted to digital images superimposed composite image Quantitative changes 43Dr Ashish U. Bisane
  • 42. • The disadvantage is the need to be almost at identical projection alignment during the exposure of sequential radiographs which makes this method impractical in clinical setting. 44Dr Ashish U. Bisane
  • 43. Diagnostic/Digital Subtraction Radiography (DSR) • Combines the use of a positioning device during film exposure with a specialized software designed for digital image subtraction using conventional personal computers in dental offices. • The software corrects the effects of angular alignment discrepancies and provides some degree of flexibility in the imaging procedure. • DSR showed statistically significant gains in diagnostic accuracy over conventional radiographs but no differences with Subtraction radiography. (Nummikoski PV, 2000) 45Dr Ashish U. Bisane
  • 44. Computer Assisted Densitometric Image Analysis System (CADIA) • A video camera measures the light transmitted through the radiograph and signals from the camera are converted into gray scale images (Bragger U, 1988) • The camera is interfaced with an image processor and a computer that allows the storage and mathematic manipulation of the images. • CADIA offers an objective method for following alveolar bone density changes quantitatively over time. • It has also shown higher sensitivity and a higher degree of reproducibility and accuracy than DSR. 46Dr Ashish U. Bisane
  • 46. • Despite difficulty in identifying all the members of the oral microbiota and understanding how they interact with each other and with the host, a limited number of micro-organisms have demonstrated a clear etiologic role and have been identified as periodontal pathogens. (Genco RJ, 1996) • Microbiologic tests to identify the putative organisms have the potential to support the diagnosis of 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. • Microbiologic tests can also be used to monitor periodontal therapy directed at the suppression or eradication of periodontal pathogenic micro-organisms.(Van Winkelhoff AJ, 1996) 48Dr Ashish U. Bisane
  • 47. Bacterial Culturing • Gold Standard • Plaque samples are cultivated under anaerobic conditions using selective and non-selective media. • Advantage: 1. Relative and Absolute count of the cultured species. • Disadvantage: 1. Strict sampling conditions 2. Difficulty in culturing most organisms 3. Low sensitivity : organisms lesser then 103 is difficult to detect 4. Time consuming 5. Expensive equipment and experienced personnel 49Dr Ashish U. Bisane
  • 48. Direct Microscopy • Dark field or phase contrast microscopy. • Most of the periodontal pathogens are non- motile so it is difficult to identify. • Even different species of Treponema cannot be distinguished by direct microscopic techniques. 50Dr Ashish U. Bisane
  • 49. Immunodiagnostic Methods • Immunologic assays employ antibodies that recognize specific bacterial antigens to detect target micro-organisms. 1. Direct and Indirect Immunofluoroscent (microscopic) Assays 2. Flow Cytometry 3. Enzyme Linked Immunosorbent Assay (ELISA) 4. Latex Agglutination 51Dr Ashish U. Bisane
  • 50. Direct Immunofluoroscent Assay (DFA) AB conjugated with Fluorescein marker Bacteria (Antigen) Immunofluoroscent complex detectable under microscope 52Dr Ashish U. Bisane
  • 52. Direct IFA Indirect IFA 54Dr Ashish U. Bisane
  • 53. Flow Cytometry or Cytofluorography Bacterial cells + species specific antibody + Secondary Fluorescein Conjugated antibody Introduced in flowcytometer. Bacterial cells are separated into single cell suspension by means of laminar flow. Cells identified by lasers. 55Dr Ashish U. Bisane
  • 54. ELISA • Similar to Antibody and Antigen reaction, but the fluorescence, instead of a radioisotope, is read using an enzymatically driven color reaction with a photometer. • Intensity of color depends upon the concentration of the antigen and is usually read photometrically for optimal quantification.  Evalusite: commercially available chairside diagnostic kit to detect Aa, Pg and Pi.  It involves linkage between antigen and a membrane bound antibody to form an immunocomplex that is revealed later with a colorimetric reaction. 56Dr Ashish U. Bisane
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  • 57. Latex Agglutination • Based on binding of protein to latex. Latex beads coated with species specific antibody When beads come in contact with specific species in sample they bind and agglutination occurs Clumping of beads is visible Test positive 59Dr Ashish U. Bisane
  • 58. Enzymatic Methods • Bacteria release specific enzymes. • Certain group of species share common enzymatic profile. • E.g. Tf, PG, Td, and Capnocytophaga species release trypsin-like enzyme. • The activity of this enzyme is measured with the hydrolysis of the colorless substrate:  N-benzoyl-d L-arginine-2-naphthylamide (BANA) 60Dr Ashish U. Bisane
  • 59. BANA Test BANA Substrate Hydrolysis by Trypsin like enzyme of bacteria Hydrolysis Formation of Chromophore: B- naphthylamide Addition of Fast Garnet The chromophore turns orange red 61Dr Ashish U. Bisane
  • 60. Perioscan is a popular diagnostic kit uses BANA reaction. Disadvantages: May be positive in clinically healthy site Cannot detect disease activity Limited organisms detected Other pathogens may be present if it’s negative. 62Dr Ashish U. Bisane
  • 61. Molecular Biology for Diagnosis • Based on analysis of DNA, RNA and structure & function of Protein of the micro-organisms. • A “probe” is a known nucleic acid molecule (DNA or RNA) from a specific micro- organism artificially synthesized and labeled for its detection when placed with a plaque sample. 63Dr Ashish U. Bisane
  • 62. Nucleic Acid Probes • A DNA probe uses a segment of a single stranded DNA, labeled with a enzyme of a radio isotope, that is able to hybridize to a complimentary nuclei strand, and thus detect presence of target microorganism. • Two types: 1. Whole genomic probes 2. Oligonucleotide probes 64Dr Ashish U. Bisane
  • 63. Whole Genomic Probes • Targets the whole DNA strand rather then a specific sequence or gene. • High chances to cross react with non target microorganism • Lower sensitivity and specificity. • Whole genomic probes for Aa, Pg, Pi and Td have been commercially developed and tested E.g.DMDx, Omnigene. 65Dr Ashish U. Bisane
  • 64. 66Dr Ashish U. Bisane
  • 65. Oligonucleotide Probes • Target variable region of 16sRNA or a specific sequence in the DNA strand. • Higher sensitivity and specificity. • These bacterial 16s RNA genes contain both regions shared by different bacteria and short stretches of variable regions shared by only specific micro-organisms of the same species or genus. (Moncla BJ, 1990) 67Dr Ashish U. Bisane
  • 66. Hybridization • Pairing of complementary strands of DNA (nucleic acid) to produce a double stranded DNA (nucleic acid). • All hybridization methods use radiolabeled or fluoroscence-labeled DNA probes that bind to the target DNA of interest, thus allowing its visualization. 68Dr Ashish U. Bisane
  • 67. Checkerboard DNA-DNA Hybridization Technology • Developed by Socransky et.al. 1994. • 40 bacterial species can be detected using whole genomic digoxigenin-labeled DNA probes. • Large number of samples can be tested and up to 40 oral species detected with a single test. • Highly specific test. • Requires expensive sophisticated laboratory equipment. 69Dr Ashish U. Bisane
  • 68. • Advantages of DNA probes as compared to bacterial culturing. 1. More sensitive and specific 2. Requires as less as 104 cells of each species to be detected. 3. Multiple species detected with a single test 4. Does not require viable bacteria 5. Large number of samples can be assessed. • Disadvantages: 1. Expensive 2. Expert personnel to carry out the test 3. Not easily available 70Dr Ashish U. Bisane
  • 69. Polymerase Chain Reaction • Developed in 1985. • Used almost universally to study DNA or RNA obtained from a variety of tissue sources. • Involves amplification of a region of DNA by a primer specific to the target species. • If there is amplification then it indicates the presence of the target species in the sample. 71Dr Ashish U. Bisane
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  • 71.  Isolation of DNA from a fresh tissue specimen.  Heating of complementary double strands of DNA.  DNA splits into single stranded forms.  Single stranded forms are intended to act as template dictating the nucleotide sequencing in vitro.  Amplification is done using a DNA polymerase that requires a primer or a known short oligonucleotide sequence complementary to the border of the region that is to be amplified.  To obtain amplified fragments of constant length and in large quantities, a second primer, complementary of the opposed chain must be used to bind the template and flank the region of interest.  This amplification can be performed several number of times known as cycles.  This sequenced DNA is then detected and visualized through electrophoresis in aragose gel and ethidium-bromure obtaining a qualitative signal. 73Dr Ashish U. Bisane
  • 72. • In 1988, a thermo-stable DNA polymerase isolated from the organism Thermus aquaticus , known as Taq-polymerase was developed. • This Taq-polymerase has allowed automatization of the reaction using specific appliances called thermocycles. 74Dr Ashish U. Bisane
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  • 74. Real Time PCRs • To quantitatively assess target bacteria using PCR technique. • By using a single copy of the bacterial genes per cell, a good correlation between the fluorescent signal measured and the number of cells can be obtained. (Lyons SR,2000; Smith AJ, 1996) 76Dr Ashish U. Bisane
  • 75. • Advantages: 1. High degree of sensitivity and specificity 2. Reproducible and consistent 3. High detection limit. As less as 5- 10 cells can be amplified and detected. 4. Less cross reactivity under optimal conditions. 5. Many species can be detected simultaneously. • Disadvantage: 1. Expensive lab equipments 2. Small quantity needed for reaction may not contain the necessary target DNA. 3. Plaque may contain enzymes which may inhibit these reactions. 77Dr Ashish U. Bisane
  • 76. Advances in Characterizing Host Response • Asses host response by studying mediators as a response to specific bacteria or local release of inflammatory mediators or enzymes as response to infection. • Source of samples may be; GCF, Saliva, or Blood. • GCF is most commonly used, where as saliva is been recently been researched recently. 1. Host derived enzymes 2. Inflammatory mediators and products 3. Tissue Breakdown Products 78Dr Ashish U. Bisane
  • 77. GCF • Most well studied, with almost 40 components in form of host-derived enzymes, tissue breakdown products, and inflammatory mediators. • Collected with paper strips, micro papillary tubes, micropipettes, microsyringes, plastic strips. • Paper strips commonly used, introduced in sulcus for 30 secs and volume is measured using Periotron 6000. • Periotron measures the capacitance across the wet paper strip, which is converted to digital reading. • Periotron reading have high correlation with clinical gingival indices. • Quickest and easiest way to measure GCF. 79Dr Ashish U. Bisane
  • 78. Perio Paper Strips Periotron 8000 80Dr Ashish U. Bisane
  • 79. Saliva • The next most used after GCF. • Easily collected • Contains both local and systemic derived markers for periodontal diseases. • Collected from parotid, sub-mandibular or sub lingual or as ‘Whole saliva’. • Whole saliva contains secretions of major and minor salivary glands, desquamated cells, and GCF. • Markers to look for in saliva: 1. Proteins and enzymes from host, 2. Phenotypic markers, 3. Host cells, 4. Hormones, 5. Bacteria, bacterial products, 6. Volatile compounds, and ions. 81Dr Ashish U. Bisane
  • 80. Cytokines • Are substances released by cells of the immune system. • Cytokines in GCF are: TNF-alpha, IL-1, IL-6, and IL-8 • Can be used to determine the disease activity. • Esp. Prostaglandin E in increased in GCF of periodontitis patients. • Can be used to determine disease activity 82Dr Ashish U. Bisane
  • 81. Host derived enzymes 1. Aspartate aminotrasnferase( AST), 2. Alkaline phosphatase, 3. B-glucoronidase, 4. Elastase, 5. Cathespins, 6. MMPs. 83Dr Ashish U. Bisane
  • 82. AST • Derived from dead cells,. • Elevated in periodontal disease • Periogard is a commercially available colorimetric test for AST. • It involves GCF collection with a filter paper strip, which is then placed in a tromethamine hydrochloride buffer. A substrate reaction mixture containing L-aspartic and a- ketoglutaric acids are added and allowed to react for 10min. In presence of AST, aspartate and glutarate are catalyzed to oxaloacetate and glutamate. Addition of a dye (Fast Red) results in a colour product. The intensity of which is proportional to the AST activity in GCF sample. • However, AST levels cannot differentiate active and inactive sites. 84Dr Ashish U. Bisane
  • 83. • ALP: released from osteoblast, neutrophils, fibroblast.. • Beta Glucoronidase and Elastase: found in Neutrophils. • Beta Glucoronidase, Elastase, Neutral protease, and Cathepsins: All are shown to be higher in diseased sites. May be used to predict severity of disease or to predict disease activity. 85Dr Ashish U. Bisane
  • 84. Matrix metalloproteinases • zinc and calcium dependent enzymes • constitutively formed in the body, secreted by fibroblast and macrophages. • Normally help in degrading and remodeling of extracellular matrices. • In chronic periodontitis they cause the degradation of the collagen fibrils in PDL and Alveolar bone. • MMP,2,3,8 9 and 13 play important role. • MMP8 level is associated with the attachment loss • Level reduces in response to treatment. (Chair side test kit) • Can be used to indicate present disease status and predictor of future disease. 86Dr Ashish U. Bisane
  • 85. Tissue Breakdown Products • Analysis of GCF obtained from sites with periodontitis clearly show elevated levels of hydroxyproline from collagen breakdown and GAGs from matrix degradation. (Last KS, 1985) • Osteocalcin and Type-I collagen peptides. 87Dr Ashish U. Bisane
  • 86. Conclusion • No proven marker available to predict the disease activity as there is no proven correlation between these markers and the clinical loss of attachment. • Search of tool with high predictive value, simple, safe and cost effective still needed. 88Dr Ashish U. Bisane
  • 87. Thank You !! 89Dr Ashish U. Bisane