Advanced diagnostic aids 24 9-12

Dr Ashish U. Bisane 1GOOD MORNING

2

Advanced Diagnostic Aids

By: Dr Ashish U. Bisane

Dr 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.


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)


• 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


• 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)


• 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).


(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.


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.


Perio-Temp


• 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.


• 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.


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.


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)


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)


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)



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.


Florida Probe System• Gibbs et al - developed Florida Probe System (Florida Probe

Corporation)

• Parts of Florida Probe System:1. Probe handpiece 2. Digital readout3. Foot switch 4. Computer interface5. 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.


Advantages

1. Constant probing force

2. Precise electronic measurement

3. Computer storage of data

Disadvantages

4. Lack of tactile sensitivity - independent movement, which forces

the operator to predetermine an insertion point and angle.

5. Use of a fixed force setting throughout the mouth, regardless of the

site or inflammatory status, may generate inaccurate measurements

or patient discomfort.

6. 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)




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.



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)


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.


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


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.



Advances in Radiographic Assessment


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.


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)


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.


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.


• 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.


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


• 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)


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.


Advantages:High correlation between alveolar bone loss

and CAL changesIncreased detection of small osseous lesionBoth quantitative and qualitative visualizationMore sensitive


• 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)


Serial radiographs

superimposed

composite image

Quantitative changes


• 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.


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)


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.


Advances in Microbiological

Analysis


• 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)


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


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.


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


Direct Immunofluoroscent Assay (DFA)

AB conjugated with Fluorescein marker

Bacteria (Antigen)

Immunofluoroscent complex detectable under microscope


Indirect Immunofluoroscent Assay

Primar

y AB

Bacteria

l AG

Immune

Complex

Secondary Fluoroscein conjugated Antibody Stains the primary AG:AB complex


Direct IFA Indirect IFA


Flow Cytometry or Cytofluorography

Bacterial cells + species specific antibody

+ Secondary Fluorescein Conjugated antibody

Introduced in flowcytometer.

Cells identified by lasers.


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.




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


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)


BANA TestBANA

Substrate

Hydrolysis by Trypsin like

enzyme of bacteria

Hydrolysis

Formation of Chromophore:

B-naphthylamide

Addition of Fast Garnet

The chromophore turns orange red


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.


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.


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


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.



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)


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.


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.


• 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


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.



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.


• 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.



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)


• 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.


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


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.


Perio Paper Strips

Periotron 8000


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.


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


Host derived enzymes

1. Aspartate aminotrasnferase( AST),

2. Alkaline phosphatase,

3. B-glucoronidase,

4. Elastase,

5. Cathespins,

6. MMPs.


AST

• Derived from dead cells,.• Elevated in periodontal disease• P e r i o g a r d 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.


• 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.


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.


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.


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.


Thank You !!

Documents

Advanced diagnostic aids 24 9-12