SACHIN RATHOD

DEPARTMENT OF PERIODONTICSDEPARTMENT OF PERIODONTICS

College Of Dental Science & Hospital Rau, Indore

LIMITATIONS OF CONVENTIONAL PERIODONTAL DIAGNOSISLIMITATIONS OF CONVENTIONAL PERIODONTAL DIAGNOSIS Advances in Clinical Diagnosis Gingival Bleeding Gingival Temperature Periodontal Probing

ADVANCES IN RADIOGRAPHIC ASSESSMENTADVANCES IN RADIOGRAPHIC ASSESSMENT Digital Radiography Subtraction Radiography Computer-Assisted Densitometric Image Analysis System

ADVANCES MICROBIOLOGIC ANALYSISADVANCES MICROBIOLOGIC ANALYSIS Bacterial Culturing Direct Microscopy Immunodiagnostic Methods Enzymatic Methods Diagnostic Assays Based on Molecular Biology Techniques

ADVANCES IN CHARACTERIZING THE HOST RESPONSEADVANCES IN CHARACTERIZING THE HOST RESPONSE Source of Samples Inflammatory Mediators and Products Host-Derived Enzymes Tissue Breakdown Products

• Periodontal disease are conventionally diagnosed by clinical evaluation of signs of inflammation in gingiva without periodontal tissue destruction (gingivitis) or by presence of both inflammation and tissue destruction(periodontitis)

• Traditional clinical diagnosis of periodontitis is made by measuring either the loss of connective tissue attachment to root surface ,i.e. clinical attachment loss or the loss of alveolar bone ,i.e. radiographic loss.

• Disease evaluation attempts to identify and quantify the current clinical signs of inflammation as well as historical evidence of damage with its extent and severity .

• However , evaluation can not reliably identify sites with ongoing periodontal destruction and does not provide inflammation on cause of condition, patient’s susceptibility to disease whether it is in progression or remission or whether the response to therapy will be positive or negative .

• Disease susceptibility is related to the whole person rather than local site .

• However periodontal disease is considered to be site specific ,it has multifactorial origin in which periodontal pathogens , host response and genetic systemic and behavioral risk factors play role in development of disease.

• Therefore consideration should be given to include microbiologic, immunologic, systemic, genetic and behavioral factors in addition to traditional clinical and radiographic parameters when assessing patient status.

Gingival Bleeding• Clinical evaluation of degree of gingival inflammation

includes assessment of redness and swelling of gingiva along with assessment of gingival bleeding.

• Gingival bleeding is related to persistent presence of plaque on teeth and regarded as a sign of associated inflammatory response.

• Use of gingival bleeding as an indicator of inflammation has the clinical advantage of being more objective , because color changes requires subjective estimation.

• Gingival bleeding is good indicator of presence of inflammatory lesion in connective tissue at the base of sulcus. The severity of bleeding increases with an increase in size of inflammatory infiltrate.

• Periodontal probe or a wooden interdental cleaner is used to elicit gingival bleeding .

• Besides an indicator of gingival inflammation gingival bleeding is also an indicator of disease activity, however its relationship to disease progression is unclear.

• Limitation of the use of bleeding as an inflammatory parameter is the possibility that healthy sites may bleed on probing, any force greater than 0.25 N may evoke bleeding in healthy sites with an intact periodontium.

• Absence of bleeding on probing indicates periodontal stability with high probability

• Tobacco smoking may mask the inflammatory signs of gingivitis and periodontitis, particularly the propensity of gingiva to bleed on brushing, eating or after periodontal probing .

• Mechanism by which smoking may exert a suppressive action on the bleeding responsiveness of the gingivae are not well understood.

• Thermal probes are sensitive diagnostic devices for measuring early inflammatory changes in gingival tissue.

• Commercially available system periotemp probe(abiodent) , enables the calculation of temperature differential(with the sensitivity of 0.1degree C) between the probed pocket and subgingival temperature.

• Individual temperature differences are compared with those expected for each tooth and higher temperature pockets are signaled with a red emitting diode.

• Subgingival temperature at diseased sites is increased as compared to normal healthy sites.

• Posterior sites are warmer than anterior region.

• Mandibular sites are warmer than maxillary sites.

• Reason why temperature increases with probing depth remains unknown .A possible explanation is an increase in cellular and molecular activity caused by increased periodontal inflammation with increasing probing depth.

• Variation in subgingival and sublingual temperature exists in smokers compared to non smokers.

Periodontal probing: The most widely used diagnostic tool for the clinical assessment of connective tissue destruction in periodontitis is the periodontal probe.Gold standard is recording changes over period of time.

Classification of periodontal probes depending

on generation.1.First generation probes:(conventional probes)Conventional manual probes that do not control for probing force or pressure and that are not suited for automatic data collection.eg: Williams periodontal probe CPITN probe UNC-15 probe University of Michigan’O’ probe Goldman Fox probe Glickman probe Merritt A and B probe Nabers probe

2.Second generation probe:(Constant force probe)Introduction of constant force or pressuresensitive probes allowed for improved standardization of probing.e.g.: Pressure sensitive probe Constant pressure probe3.Third generation probe:(Automated probes)Computer assisted direct data capture was an important step in reducing examiner bias and also allowed for generation probe precision.e.g.: Toronto probe Florida probe Interprobe, Foster Miller probe.

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 andThree dimensional pocket that is being examined.5.Fifth generation probe:(Noninvasive)Three dimensional probe.Basically these will add an ultrasound or anotherdevice 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.

~:Problems with probing:

Lack of sensitivity and reproducibility.Readings of clinical pocket depth obtained with periodontal probe do not normally coincide with the histologic pocket depth because the probe normally penetrates the coronal level of the junctional epithelium.

~:Probing depends on: force, angulations, size of

probe, precision of calibration presence of inflammation. All these variables contributes to large standard deviations(0.5-1.3) in clinical probing result

{force to probe pocket: 30g} {force to probe periodontal osseous defect:50g}

Since mid 1980s different probe prototypes

are developed & tested to overcome these limitations. One of the problem was of improper penetration of probe related to improper force of probing which was solved with the development of pressure sensitive probes which have

standardized, controlled insertion pressure.~New technology n high tech computers

are nowadays becoing the rule. It has provided to dentists ideal potential of standardization which makes future comparison of health & disease simpler, more precise and cost effective.

Disadvantages of Florida probe.

Lack of tactile sensitivity because of their independent movement.

Fixed probing force Underestimation of deep periodontal pockets.

Other electronic probes:

Improvised Florida PASHA probeInterprobePerioprobeFoster Miller probeToronto Automate ( difficult to reproduce patient

head position and in 2nd and 3rd Molar area.)

Dental Radiographs are traditionally used to access destruction of alveolar bone associated with periodontitis. They provide information on the periodontium that cannot be obtained by other noninvasive methods.Problems with conventional Radiography: Variation in projection geometry Variation in contrast and density Masking by other anatomic structures. They are very specific but lack

sensitivity.

Substraction Radiography:

Serial radiographs converted to digital images superimposed composite image– Quantitative changes

Limitations: needs paralleling technique and accurate superimposition.

Advantages:

High correlation between alveolar bone loss and CAL changes

Increased detection of small osseous lesion Both quantitative and qualitative visualization More sensitive

Disadvantage: Identical projection alignment during

sequential radiographs.

Diagnostic SR: uses position device for films and

software that corrects angular alignment discrepancy.

Computer Assisted Densitometric Image Analysis. (CADIA)

Video camera measures the light transmitted through radiograph and the signals form the camera is converted to gray scale image.

Advantage: Measures quantitative changes in bone density overtime.Higher sensitivity, reproducibility and

accuracy as compared to DSR.

Computed Tomography scanning is widely used in the evaluation of the implant patient.

HOWS IT WORK?

A thin fan beam of X-Rays rotates around the patient to generate in one resolution a thin axial slice of the area of interest.

Multiple overlapping axail sclices are obtained by several revolution of the X-ray beam until the whole area of interest is covered.

Then these slices are then used to generate with the help of a computer and sophisticated Algorithms.

A three dimensional digital map of the jaw is formed which help in evaluation of the implant patient.

Specialized software can be used to generate appropriate views that best depict the dimensions of the jaws and the location of important anatomic structures.

DENTAL VIEWS OBTAINED FROM A CT SCAN INCLUDE:-

1. AXIAL 2. PANORAMIC 3. CROSS-SECTIONAL. Views of the Jaws.

A

B

ADVANTAGES of Computed TomographyADVANTAGES of Computed Tomography

1. 1. True cross sections offer a precise and detailed evaluation of the height and width of the ALVEOLAR RIDGE.

2. The images can be adjusted and printed without magnification, facilitating measurements directly on the prints or films with standard rulers.

3. Various anatomic structures can be visualized and analyzed at all the Coordinate axis.

1. Superioinferior 2. Anteroposterior 3. Buccolingual

Images of the entire arch several edentulous areas can be visualized with single examination.

The Bone and soft tissue contrast and resolution are excellent for the diagnostic task.

DISADVANTAGES of Computed DISADVANTAGES of Computed TomographyTomography CT scanning requires specialized equipment and

setting. Radiologists and Technicians need to be

knowledgeable of the anatomy, anatomic variants and pathology of the jaws as well as considerations pertinent implant treatment planning.

CT scan delivers higher radiation dose to the patient as compared to other modalities used during implant treatment planning.

CT scan images is of whole arch, it delivers radiation to whole arch.

Metallic Restorations can cause ring artifacts that impair the diagnostic quality of the image, it is challenging to the patients having heavy metallic restored dentition.

Cone-Beam Computed Tomography (CBCT) is a new imaging modality that offers significant advantages for the evaluation of implant patients.

HOW IT WORK?

The X-Ray Source and the detector are dramatically positioned and make a 360 degree rotation around this patient head within the quantry.

In CBCT scanner generates a Cone shaped X-Ray Beam, which images larger area.

Images are generated in 1 degree increments, so at end of a single rotation 360 images of area are generated.

The computer uses these images to generate a Digital three dimensional map of the face.

Once map is generated multiplanar recounstructions as well as axail, coronal, saggital, or oblique images are generated.

11. . True cross sections offer a precise and detailed evaluation of the height and width of the ALVEOLAR RIDGE.

2. The images can be adjusted and printed without magnification, facilitating measurements directly on the prints or films with standard rulers.

3. Various anatomic structures can be visualized and analyzed at all the Coordinate axis.

1. Superioinferior 2. Anteroposterior 3. Buccolingual

Images of the entire arch several edentulous areas can be visualized with single examination.

CBCT SCANNER DELIVERS THE REDUCED AMOUNT OF RADIATION DOSE TO PATIENT THAN OTHER MODALITY.

IT IS SPECIALLY MENT IN RELATION FOR DENTAL IMPLANTS.

CBCT DELIVERS AN EFFECTIVE DOSE APPROXIMATELY EQUAL TO A FULL-MOUTH X-RAY SERIES THIS IS 50 TO 100 TIMES LESS THAN THE OTHER MODALITY.

CBCT SCANNING IS A VALUABLE IMAGING MODALITY FOR THREE-DIMENSIONAL AND CROSS-SECTIONAL EVALUATION OF THE IMPLANT PATIENT.

CBCT scanning requires specialized equipment and setting.

Radiologists and Technicians need to be knowledgeable of the anatomy, anatomic variants and pathology of the jaws as well as considerations pertinent implant treament planning.

CBCT scan delivers higher radiation dose to the patient as compared to other modalities used during implant treatment planning.

CBCT scan images is of whole arch, it delivers radiation to whole arch.

Metallic Restorations can cause ring artifacts that impair the diagnostic quality of the image, it is challenging to the patients having heavy metallic restored dentition.

CBCT SCANS HAVE A LIMITED CAPACITY TO SEPARATE MUSCLE FROM FAT OR CONNECTIVE TISSUE COMPARED WITH CT SCAN.

COMPUTED TOMOGRAPHYCOMPUTED TOMOGRAPHY CONE-BEAM COMPUTED CONE-BEAM COMPUTED TOMOGRAPHYTOMOGRAPHY

A thin fan-beam of X-Rays rotates around the patients to generate in ONE REVOLUTION A THIN AXIAL SLICE OF AREA OF INTEREST.

In one Revolution one Axial slice is formed.

A Fan Beam X-Rays are generated.

CT Scan offers a greater contrast resolution or the ability to distinguish two objects with small density differences.

The X-ray source and the Detector are diametrically positioned and make a 360 degree rotation around the patient head within the gantry.

In one 360 degree rotation 360 images are formed.

CBCT scanner generates a cone-shaped X-ray beam, forms a larger image.

But CBCT cant distinguish properly.

COMPUTED TOMOGRAPHYCOMPUTED TOMOGRAPHY CONE-BEAM COMPUTED CONE-BEAM COMPUTED TOMOGRAPHYTOMOGRAPHY

CT Scan has higher capacity to separate muscle from fat or connective tissue.

MOST SGNIFICANT DIFFERENCE

More amount of Radiation dose is delivers to patient while scanning.

It is used for Dental implants, Neurological Diagnosis, Orthopedics Diagnosis etc.

CBCT has limited capacity to separate muscle from fat or connective tissue.

MOST SIGNIFICANT DIFFERNCE

Very limited amount of Radiation dose is delivers to patient while scanning, effective dose is approximately equal to full mouth series, this is 50 to 100 times less than the radiation dose delivers to patient than CT Scan.

It is mainly used in Dental Implant purpose.

COMPUTED TOMOGRAPHYCOMPUTED TOMOGRAPHY CONE-BEAM COMPUTED CONE-BEAM COMPUTED TOMOGRAPHYTOMOGRAPHY

Subgingival microenvironment has 300+ species Only few organisms are thought to be involved

with periodontal disease. Strong evidence for actinomycetemcomitans

(Aa), Porphyromonas gingivalis (Pg), and Tannerella forsythia (Tf).

Other organisms that are thought to have etiologic role are Camphylobacter rectus, Eubaterium nodatum, Fusobacterium nucleatum, Peptostreptococcus micros, Prevetolla intermedia and Prevetolla nigrescens, Trepenoma Denticola.

Uses of microbiologic analysis Support diagnosis. Treatment planning Indicator for disease activity

( absence is a better indicator)

Plaque samples are cultivated under anaerobic conditions using selective and nonselective media.

Advantage:Relative and Absolute count of the cultured

species.Disadvantage:Strict sampling conditionsDifficulty in culturing most organismsLow sensitivity : organisms lesser then 103 is

difficult to detectTime consumingExpensive equipment and experienced personnel

P. intermedia

P. Gingivalis

P. Forsythia

F. Nucleatum

Direct Microscopy: Most of the periodontal pathogens are non motile so it is difficult to identify.

That’s why dark field microscopy seems an unlikely candidate as a diagnostic test of destructive periodontal diseases.

Immunofloresence Assay (IFA):

Direct Indirect

Direct IFA: AB conjugated with Fluorescein marker + Bacteria ( Antigen) = Immuno complex

Indirect IFA: Primary AB + Bacteria= Immune Complex+ Secondary Fl conjugated AB

Direct IFA Indirect IFA

Flow CytometryBacterial cells+ species specic AB +

Secondary FL Conjugated AB Introduced in flowcytometer

Bacterial cells is separated into single cell suspension- passes through the tube Cells identified by lasers.

Latex Agglutination TestLatex beads coated with species specific

AB when beads come in contact with specific species in sample they bind and agglutination occurs clumping of beads is visible test positive.

THIS method is not clinically available.its olny for research.

Advantages:Simple and Rapid testingHigher sensitivity and specificity.

ELISA= Enzyme Linked Immunosorbent Assay

Similar AB and Antigen reaction, but the fluorescence is read using a photometer.

Evalusite: commercially available kit to detect Aa,Pg and Pi.

Well with precoated antibody + Sample to be tested= immune complex

Specific antigen bind to the antibody + Secondary antibody added.

Immunofloresence dye bound to secondary antibody

Substrate added which changes the color of the solution

Amount of florescence checked by photometer (450nm)

SANDWICH ELISA Specific antibody ->antigenconjugate

(antibody to antigen)->substrate added ->colour (positive result)

. Perioscan is a popular diagnostic kit uses BANA reaction.

Disadvantage: May be positive in clinically healthy site

Cannot detect disease activity Limited organisms detected Other pathogens may be present if it’s

negativE

Perioscan requires a plaque sample to detect the presence of enzymes capable of degrading N-benzoyl-DL-arginine-2-naphthylamide (BANA) from relatively few anaerobic periodontal pathogens.

Basic Principle: Analysis of DNA, RNA and protein structure.

Hybridization: Pairing of complimentary strands of DNA to produce a double stranded DNA.

Nucleic acid probe: is a known DNA/RNA which is synthesized artificially and labeled with a enzyme or a radioisotope for detection when placed in a plaque sample.

DNA Structure

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.

DNA Probe

Two types of DNA probesWhole genomic: 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 GenomicDNA Probe

Oligonucleotide probes: target variable region of 16sRNA or a specific sequence in the DNA strand.

Higher sensitivity and specificity.

Checkerboard DNA-DNA Hybridization Technology:

Developed by Socransky et.al.40 bacterial species can be detected using

whole genomic digoxigenin-labeled DNA probes.

Large number of samples can be tested and upto 40 oral species detected with a single test.

Advantages of DNA probes as compared to bacterial culturing.

1. More sensitive and specific2. Requires as less as 104 cells of each species

to be detected.3. Multiple species detected with a single test4. Does not require viable bacteria5. Large number of samples can be assessed.Disadvantage:1. Expensive 2. Expert personnel to carry out the test3. Not easily available

Polymerase Chain Reaction (PCR):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.

Advantages:1. High detection limit. As less as 5- 10 cells

can be amplified and detected.2. Less cross reactivity under optimal

conditions3. Many species can be detected

simultaneouslyDisadvantage:1. Small quantity needed for reaction may not

contain the necessary target DNA2. Plaque may contain enzymes which may

inhibit these reactions.

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.

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.

GCF :-

Perio Paper StripsPeriotron 8000Periotron 8000

It is the next most used after GCFeasily collected

contain both local and systemic derived markers for periodontal disease

Collected from parotid, sub-mand or sub lingual or as ‘Whole saliva’

Whole saliva contains secretions of major and minor salivary glands, desquamated cells, and GCF.

No diagnostic test available in the market although lot of research is in progress.

Markers to look for in saliva: proteins and enzymes from host, phenotypic markers, host cells, hormones, bacteria, bacterial products, volatile compounds, and ions.

Saliva:

Cytokines: are substances released by cells of the immune system.

Cytokines in GCF are: TNF-alpha, IL-1, IL-6, and IL-8

Have actions on immune cells and release of enzymes, including bone resorption.

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

Various enzyme are released from the host cell during the initiation & progression of periodontal disease.

Matrix components may be dissolve either by extra cellular matrix metalloproteinase dependent or lysosomal proteinase.

The protease & enzyme involved in this process may have use as diagnostic aids & thus their role should be considered further.

The breakdown of collagen occurs during inflammation , remodeling & wound healing.

Two different pathway,• Intracellular• Extracellular

Under non-pathologic condition, phagocytosis & intracellular digestion of collagen fibrils is a process observed.(in gingiva & pdl)

In pathologic condition,

the balance between synthesis & degradation is disrupted

The collagen fibrils of PDL are broken down with supporting alveolar bone.

Different enzymes involved in both the intracellular & extracellular pathway of tissue destruction.

Intracellular destruction enzyme:- Aspartate aminotransferase Alkaline phosphate Beta glucoronidase Elastase

Released from dead & dying cells of PDL Mostly from PMNs ,neutrophils.

Extracellular destruction enzyme, Matrix metalloproteinases- produced

by inflammatory epithelial & connective tissue cells at affected sites.

AST: derived from dead cellsElevated in GCF in periodontal diseasePeriogard is a commercially available colorimetric test.

Collection of GCF with filterpaper strip

Placed into Tromethamine Hydrochloride buffer

Add L-aspartic & alpha-ketoglutaric acid 10 min

reaction timeIf AST present

The aspartate &glutarate catalyzed to oxalatate & glutamate

Gives red colour

A potenttial problem with the AST test is its inability to discriminate between site with severe inflammation but with no attechment loss from sites that are losing attachment.

ALP: released from osteoblast, neutrophils, fibroblast..

BG and Elastase: found in Neutophils.BG may have predictive value in patient at

higher risk for losing attachment.Cathepsins-acidic lysosomal enzyme .all shown to be higher in diseased sites.

May be used to predict severity of disease or to predict disease activity.

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

In periodontitis patient increased level of MMP-8.

Level reduces in response to treatment. (Chair side test kit)

Can be used to indicate present disease status and predictor of future disease.

One of the major feature of periodontitis is the destruction of collagen & extracellular matrices.

The connective tissue of periodontium is composed of fibrous (collagen & elastin)&non fibrous (glycoproteins),water etc.

The extracellular matrix is composed of collagen,proteoglycan & noncollagen protein.

In periodontitis elevated level of hydroxyproline from collagen breakdown &glycosaminoglycans from matrix degradation.

Conclusion:No marker available to predict the disease

activity as there is no proven correlation between these markers and the clinical loss of attachment.

In search of tool with high predictive value, simple, safe and cost effective.

What are diagnostic aids and write about advanced diagnostic aid

ELISA Test BANA Reaction Role of Saliva Advanced Diagnostic Techniques Periodontal Probes Role of Saliva in Oral Health GCF MMP B-Glucoronidase AST Enumerate Adv Diagnostic Aids and

Elaborate on DNA Probe/ Microbial Analysis/

Carranza 10 edition

SACHIN RATHODSACHIN RATHODEMAIL:- drsachin.rathod@yahoo.comEMAIL:- drsachin.rathod@yahoo.com