Advanced Advanced Diagnostic AidsDiagnostic Aids

Dr V. M. JoshiDr V. M. Joshi

Periodontitis:Periodontitis: is characterized by a is characterized by a loss of connective tissue attachment loss of connective tissue attachment that begins at, or just apical to, the that begins at, or just apical to, the cementoenamel junction and extends cementoenamel junction and extends apically along the root surface.apically along the root surface.

Disease Activity?Disease Activity?

Periodontal Disease Markers?Periodontal Disease Markers?

How are they Important??How are they Important??

Advances in Clinical diagnosisAdvances in Clinical diagnosis Advances in Radiographic Advances in Radiographic

AssessmentAssessment Advances in Microbiologic AnalysisAdvances in Microbiologic Analysis Advances in Characterizing the Host Advances in Characterizing the Host

ResponseResponse

I. Advances in Clinical I. Advances in Clinical DiagnosisDiagnosis

Gingival Bleeding:Gingival Bleeding: Indicator of inflammatory lesionIndicator of inflammatory lesion Relation to disease activity is Relation to disease activity is

unclear.unclear. Normal probing force is Normal probing force is 0.25N0.25N Presence is not a indicator but Presence is not a indicator but

absence indicates health.absence indicates health.

Gingival Temperature:Gingival Temperature: Subgingival temperature as a diagnostic tool Subgingival temperature as a diagnostic tool

(PerioTemp)(PerioTemp) Subgingiaval temperature in increased at Subgingiaval temperature in increased at

diseased sites. (increased cellular and diseased sites. (increased cellular and molecular activity)molecular activity)

Variation in natural temperature gradient.Variation in natural temperature gradient. Haffajee et.al. : attachment loss in shallow Haffajee et.al. : attachment loss in shallow

pockets and presence of Pg,Pi, Tf, Aa, Pm. pockets and presence of Pg,Pi, Tf, Aa, Pm. (relationship not know)(relationship not know)

Smokers have differences in subgingival Smokers have differences in subgingival temperature and sublingual temperature.temperature and sublingual temperature.

Periodontal probing:Periodontal probing:Gold standard is recording changes Gold standard is recording changes over period of time.over period of time.Problems with probing:Problems with probing: Lack of sensitivity and reproducibility.Lack of sensitivity and reproducibility.Probing depends on: force, angulations, size of Probing depends on: force, angulations, size of

probe, precision of calibration, presence of probe, precision of calibration, presence of inflammation.inflammation.

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

50g}50g}

Florida probe:Florida probe:

Tip is 0.4mmTip is 0.4mm Sleeve- edge provides referenceSleeve- edge provides reference

to make measurementsto make measurements Coil Spring; provides constant Coil Spring; provides constant

probing forceprobing force Computer for data storage.Computer for data storage.

FP Handpiece tip as it enters the sulcus.

FP Handpiece tip with constant force in use (tip at bottom of sulcus) and sleeve properly positioned at the top of the gingival margin allowing the computer to measure the difference (3.0 mm).

DisadvantagesDisadvantages of Florida probe. of Florida probe.Lack of tactile sensitivityLack of tactile sensitivity Fixed probing forceFixed probing force Underestimation of deep periodontal pockets.Underestimation of deep periodontal pockets.Other electronic probes:Other electronic probes:Improvised Florida PASHA probeImprovised Florida PASHA probeInterprobeInterprobePerioprobePerioprobeFoster Miller probeFoster Miller probeToronto Automate ( difficult to reproduce patient Toronto Automate ( difficult to reproduce patient

head position and in 2head position and in 2ndnd and 3 and 3rdrd Molar area.) Molar area.)

II. Advances in II. Advances in Radiographic AssessmentRadiographic Assessment

Problems with conventional Problems with conventional Radiography:Radiography:

Variation in projection geometryVariation in projection geometry Variation in contrast and densityVariation in contrast and density Masking by other anatomic structures.Masking by other anatomic structures.

Digital Radiography:Digital Radiography:

Advantage: digital storage, image Advantage: digital storage, image enhancement, and radiation dose enhancement, and radiation dose reduction.reduction.

Substraction Radiography:Substraction Radiography: Serial radiographsSerial radiographs converted to converted to

digital imagesdigital images superimposed superimposed composite image– Quantitative composite image– Quantitative changeschanges

Limitations: needs paralleling Limitations: needs paralleling technique and accurate technique and accurate superimposition.superimposition.

Advantages:Advantages: High correlation between alveolar bone loss and CAL High correlation between alveolar bone loss and CAL

changeschanges Increased detection of small osseous lesionIncreased detection of small osseous lesion Both quantitative and qualitative visualizationBoth quantitative and qualitative visualization More sensitiveMore sensitiveDisadvantage:Disadvantage: Identical projection alignment during sequential Identical projection alignment during sequential

radiographsradiographs

Digital SR:Digital SR: uses position device for films and software uses position device for films and software that corrects angular alignment discrepancy.that corrects angular alignment discrepancy.

Computer Assisted Densitometric Computer Assisted Densitometric Image Analysis. (CADIA)Image Analysis. (CADIA)

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

Advantage:Advantage: Measures quantitative changes in bone Measures quantitative changes in bone

density overtime.density overtime. Higher sensitivity, reproducibility and Higher sensitivity, reproducibility and

accuracy as compared to DSR.accuracy as compared to DSR.

III. Advances In III. Advances In Microbiologic AnalysisMicrobiologic Analysis

Subgingival microenvironment has 300+ Subgingival microenvironment has 300+ species species

Only few organisms are thought to be Only few organisms are thought to be involved with periodontal disease.involved with periodontal disease.

Strong evidence for Aa, Pg, and Tf.Strong evidence for Aa, Pg, and Tf. Other organisms that are thought to have Other organisms that are thought to have

etiologic role are Camphylobacter rectus, etiologic role are Camphylobacter rectus, Eubaterium nodatum, Fusobacterium Eubaterium nodatum, Fusobacterium nucleatum, Peptostreptococcus micros, nucleatum, Peptostreptococcus micros, Prevetolla intermedia and Prevetolla Prevetolla intermedia and Prevetolla nigrescens, Td.nigrescens, Td.

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

( absence is a better indicator)( absence is a better indicator)

Bacterial culturingBacterial culturing

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

Advantage:Advantage:Relative and Absolute count of the cultured species.Relative and Absolute count of the cultured species.Disadvantage:Disadvantage:Strict sampling conditionsStrict sampling conditionsDifficulty in culturing most organismsDifficulty in culturing most organismsLow sensitivity : organisms lesser then 10Low sensitivity : organisms lesser then 103 3 is difficult is difficult

to detectto detectTime consumingTime consumingExpensive equipment and experienced personnel Expensive equipment and experienced personnel

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

Immunodiagnostic Immunodiagnostic MethodsMethods

Immunofloresence Assay (IFA):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 CytometryFlow Cytometry

Bacterial cells+ species specic AB + Bacterial cells+ species specic AB + Secondary FL Conjugated ABSecondary FL Conjugated AB Introduced in flowcytometerIntroduced in flowcytometer

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

ELISA= Enzyme Linked ELISA= Enzyme Linked Immunosorbent AssayImmunosorbent Assay

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

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

Latex Agglutination TestLatex Agglutination TestLatex beads coated with species Latex beads coated with species

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

Advantages:Advantages:Simple and Rapid testingSimple and Rapid testingHigher sensitivity and specificity.Higher sensitivity and specificity.

Enzymatic MethodsEnzymatic Methods

Bacteria release specific enzymes. Bacteria release specific enzymes. Certain group of species share common Certain group of species share common enzymatic profile.enzymatic profile.

e.g. Tf, PG, Td, and Capnocytophaga e.g. Tf, PG, Td, and Capnocytophaga species release trypsinlike enzyme.species release trypsinlike enzyme.

Enyme hydrolysis specific substrate Enyme hydrolysis specific substrate (BANA)(BANA) release cromophore ( B- release cromophore ( B-naphtalamide)naphtalamide) Cromophore changes Cromophore changes color on addition of a substance( fast color on addition of a substance( fast garnet)garnet)

Perioscan is a popular diagnostic kit uses Perioscan is a popular diagnostic kit uses BANA reaction.BANA reaction.

Disadvantage:Disadvantage:

May be positive in clinically healthy May be positive in clinically healthy sitesite

Cannot detect disease activityCannot detect disease activity

Limited organisms detectedLimited organisms detected

Other pathogens may be present if it’s Other pathogens may be present if it’s negative.negative.

Molecular Biology Molecular Biology TechniquesTechniques

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

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

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

DNA Structure

DNA Probe:DNA Probe: uses a segment of a uses a segment of a single stranded DNA, labeled with a single stranded DNA, labeled with a enzyme of a radio isotope, that is enzyme of a radio isotope, that is able to hybridize to a complimentary able to hybridize to a complimentary nuclei strand, and thus detect nuclei strand, and thus detect presence of target microorganism.presence of target microorganism.

DNA ProbeDNA Probe

DNA DNA ProbeProbe

Two types of DNA probesTwo types of DNA probes

Whole genomic:Whole genomic: Targets the whole Targets the whole DNA strand rather then a specific DNA strand rather then a specific sequence or gene.sequence or gene.

High chances to cross react with non High chances to cross react with non target microorganismtarget microorganism

Lower sensitivity and specificity.Lower sensitivity and specificity.

Whole GenomicWhole Genomic

DNA ProbeDNA Probe

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

Higher sensitivity and specificity.Higher sensitivity and specificity.

Oligonucleotide Oligonucleotide probesprobes

Checkerboard DNA-DNA Checkerboard DNA-DNA Hybridization Technology:Hybridization Technology:

Developed by Socransky et.al.Developed by Socransky et.al.

40 bacterial species can be detected 40 bacterial species can be detected using whole genomic digoxigenin-using whole genomic digoxigenin-labeled DNA probes.labeled DNA probes.

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

Advantages of DNA probesAdvantages of DNA probes as compared to as compared to bacterial culturing.bacterial culturing.

1.1. More sensitive and specificMore sensitive and specific2.2. Requires as less as 10Requires as less as 1044 cells of each species to cells of each species to

be detected.be detected.3.3. Multiple species detected with a single testMultiple species detected with a single test4.4. Does not require viable bacteriaDoes not require viable bacteria5.5. Large number of samples can be assessed.Large number of samples can be assessed.Disadvantage:Disadvantage:1.1. Expensive Expensive 2.2. Expert personnel to carry out the testExpert personnel to carry out the test3.3. Not easily availableNot easily available

Polymerase Chain Reaction Polymerase Chain Reaction (PCR):(PCR):

Involves amplification of a region of Involves amplification of a region of DNA by a primer specific to the DNA by a primer specific to the target species.target species.

If there is amplification then it If there is amplification then it indicates the presence of the target indicates the presence of the target species in the sample.species in the sample.

Advantages:Advantages:1.1. High detection limit. As less as 5- 10 cells High detection limit. As less as 5- 10 cells

can be amplified and detected.can be amplified and detected.2.2. Less cross reactivity under optimal Less cross reactivity under optimal

conditionsconditions3.3. Many species can be detected simultaneouslyMany species can be detected simultaneouslyDisadvantage:Disadvantage:1.1. Small quantity needed for reaction may not Small quantity needed for reaction may not

contain the necessary target DNAcontain the necessary target DNA2.2. Plaque may contain enzymes which may Plaque may contain enzymes which may

inhibit these reactions.inhibit these reactions.

IV. Advances in IV. Advances in Characterizing Host Characterizing Host

ResponseResponse Asses host response by studying Asses host response by studying

mediators as a response to specific mediators as a response to specific bacteria or local release of inflammatory bacteria or local release of inflammatory mediators or enzymes as response to mediators or enzymes as response to infection.infection.

Source of samples may be; GCF, Saliva, Source of samples may be; GCF, Saliva, or Blood.or Blood.

GCF is most commonly used, where as GCF is most commonly used, where as saliva is been recently been researched saliva is been recently been researched recently.recently.

GCF:GCF:Most well studied, with almost 40 components in Most well studied, with almost 40 components in

form of host-derived enzymes, tissue breakdown form of host-derived enzymes, tissue breakdown products, and inflammatory mediators.products, and inflammatory mediators.

Collected with paper strips, micro papillary tubes, Collected with paper strips, micro papillary tubes, micropipettes, microsyringes, plastic strips.micropipettes, microsyringes, plastic strips.

Paper strips commonly used, introduced in sulcus Paper strips commonly used, introduced in sulcus for 30 secs and volume is measured using for 30 secs and volume is measured using Periotron 6000.Periotron 6000.

Periotron measures the capacitance across the wet Periotron measures the capacitance across the wet paper strip, which is converted to digital reading.paper strip, which is converted to digital reading.

Periotron reading have high correlation with Periotron reading have high correlation with clinical gingival indices.clinical gingival indices.

Quickest and easiest way to measure GCF.Quickest and easiest way to measure GCF.

Perio Paper StripsPeriotron 8000Periotron 8000

Saliva: is the next most used after GCFSaliva: is the next most used after GCFeasily collectedeasily collectedcontain both local and systemic derived markers for contain both local and systemic derived markers for

periodontal diseaseperiodontal disease Collected from parotid, sub-mand or sub lingual or Collected from parotid, sub-mand or sub lingual or

as ‘Whole saliva’as ‘Whole saliva’ Whole saliva contains secretions of major and Whole saliva contains secretions of major and

minor salivary glands, desaqumated cells, and GCF.minor salivary glands, desaqumated cells, and GCF. No diagnostic test available in the market although No diagnostic test available in the market although

lot of research is in progress.lot of research is in progress. Markers to look for in saliva: proteins and enzymes Markers to look for in saliva: proteins and enzymes

from host, phenotypic markers, host cells, from host, phenotypic markers, host cells, hormones, bacteria, bacterial products, volatile hormones, bacteria, bacterial products, volatile compounds, and ions.compounds, and ions.

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

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

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

Can be used to determine the disease activity.Can be used to determine the disease activity.

Esp. Prostaglandin E in increased in GCF of Esp. Prostaglandin E in increased in GCF of periodontitis patients.periodontitis patients.

Can be used to determine disease activityCan be used to determine disease activity

Host derived enzymes:Host derived enzymes:Aspartate aminotrasnferase( AST),Alkaline Aspartate aminotrasnferase( AST),Alkaline

phosphatase, B-glucoronidase, elastase, phosphatase, B-glucoronidase, elastase, cathespins, and MMPs.cathespins, and MMPs.

AST: derived from dead cellsAST: derived from dead cellsElevated in periodontal diseaseElevated in periodontal diseasePeriogard is a commercially available Periogard is a commercially available

colorimetric test.colorimetric test.Cannot differentiate active and inactive Cannot differentiate active and inactive

sites.sites.

ALP: released from osteoblast, ALP: released from osteoblast, neutrophils, fibroblast..neutrophils, fibroblast..

BG and Elastase: found in Neutophils.BG and Elastase: found in Neutophils.

BG, Elastase, Neutral protease, and BG, Elastase, Neutral protease, and cathepsinscathepsins

all shown to be higher in diseased all shown to be higher in diseased sites. May be used to predict severity sites. May be used to predict severity of disease or to predict disease of disease or to predict disease activity.activity.

Matrix metalloprotienases:Matrix metalloprotienases:zinc and calcium dependent enzymeszinc and calcium dependent enzymesconstitutively formed in the body, secreted by constitutively formed in the body, secreted by

fibroblast and macrophages.fibroblast and macrophages.Normally help in degrading and remodeling of Normally help in degrading and remodeling of

extracellular matrices.extracellular matrices.In chronic periodontitis they cause the degradation In chronic periodontitis they cause the degradation

of the collagen fibrils in PDL and Alveolar bone.of the collagen fibrils in PDL and Alveolar bone.MMP,2,3,8 9 and 13 play important role.MMP,2,3,8 9 and 13 play important role.MMP8 level is associated with the attachment lossMMP8 level is associated with the attachment lossLevel reduces in response to treatment. (Chair side Level reduces in response to treatment. (Chair side

test kit)test kit)Can be used to indicate present disaese status and Can be used to indicate present disaese status and

predictor of future disease.predictor of future disease.

Conclusion:Conclusion:

No marker available to predict the No marker available to predict the disease activity as there is no proven disease activity as there is no proven correlation between these markers correlation between these markers and the clinical loss of attachment.and the clinical loss of attachment.

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

Exam Point of ViewExam Point of View

What are diagnostic aids and write about advanced What are diagnostic aids and write about advanced diagnostic aiddiagnostic aid

ELISA TestELISA Test Diagnostic Aids Including Rotengenography its uses and Diagnostic Aids Including Rotengenography its uses and

limitationslimitations Role of SalivaRole of Saliva Advanced Diagnostic TechniquesAdvanced Diagnostic Techniques Periodontal ProbesPeriodontal Probes Role of Saliva in Oral HealthRole of Saliva in Oral Health GCFGCF MMPMMP B-GlucoronidaseB-Glucoronidase ASTAST Enumerate Adv Diagnostic Aids and Elaborate on DNA Enumerate Adv Diagnostic Aids and Elaborate on DNA

Probe/ Microbial Analysis/ Probe/ Microbial Analysis/

Recommended ReadingRecommended Reading

Carranza 10 editionCarranza 10 edition Testing for Marker Bacteria In Testing for Marker Bacteria In

Progressive Periodontitis: The Progressive Periodontitis: The European Experience, European Experience, Infectious Diseases in Infectious Diseases in Clinical Practice: December 2001 - Volume 10 - Issue 9 - Clinical Practice: December 2001 - Volume 10 - Issue 9 - pp 481-487 Mini Reviewpp 481-487 Mini Review