Advanced Diagnostic Aids

Dr. Khushbu mishraHKE’s S.N Dental college,

Gulbarga

Contents

Introduction Limitations of conventional periodontal diagnosis Advances In Clinical Diagnosis Advances In Radiographic Assessment Advances Microbiologic Analysis Advances In Characterizing The Host Response

Introduction

• Definition : Diagnosis is defined as identifying the disease from an evaluation of history, signs and symptoms, laboratory tests and procedures.

• Importance :

a. It identifies and indicates the nature of etiological factors

b. Indicates the nature of pathological processes

c. It is essential for treatment planning

types of diagnosis

Provisional differential comprehensive

therapeutic

emergency

Diagnostic aids in periodontics conventional

advanced

• Periodontal disease Gingival Inflammation

Periodontal Destruction

• Diagnosis :- clinical evaluation ( Gingivitis)

clinical evaluation

+ (periodontitis)

tissue destruction

• Tissue destruction ( Periodontitis)

Loss of connective tissue

clinical radiographic

attatchment loss bone loss

Gives historical evidence of damage Identify and quantify current clinical signs of inflammation

Limitations

• Does not provide cause of condition• Susceptibility of patient• Cannot reliably identify sites with ongoing periodontal

destruction• Cannot differentiate whether response to therapy is positive or

negative

• Periodontal disease is localised and multifactorial.

Periodontal

pathogens

Host response

behaviouralsystemic

Genetic

• Consideration should be given microbiologic

immunologic

systemic

genetic

behavioural factors

in addition to clinical and radiographic parameters.

• The focus is now disease prevention, early discovery, and intervention to minimize treatment, thus enabling the most desirable outcomes.

• Diagnostic modalities available to clinicians today expand greatly on the foundation of a comprehensive visual assessment, which has been and will be the cornerstone of the diagnostic process.

Classification

• Aids used in clinical diagnosis

i. Conventional probes – regular examination

ii. Millimeter probes – for gingival bleeding

iii. Pressure sensitive probes

Other clinical diagnostic aids

i. Filter papers- for measuring GCF

ii. Periotron 6000- for measuring GCF

iii. Olfactometer- for mouth odors

iv. Mobilometer- for tooth mobility

v. PSR- for faster screening and recording of PD

• Aids used in radiographic diagnosis

i. IOPA radiographs

ii. Ortho-pantomograph

iii. Xero-radiography

• Aids used in microbial diagnosis

i. Direct examination

a. Light microscopy

b. Dark field microscopy

ii. Culture tests

a. Aerobic culture

b. Anaerobic culture

• Aids used in immunological diagnosis

i. Immunofluorescense- direct and indirect

ii. Polymerase chain reaction

iii. Latex agglutination

iv. Flow cytometry

v. ELISA• Biochemical diagnosis

i. Studies for prostaglandins

ii. Studies for collagenase• Other diagnostic aids

i. study casts

ii. FSEIA

iii. N-benzoyl-DL- arginine 2- naphthylamide (BANA)

Advanced diagnostic aids

• Advanced periodontal probes

i. Automated controlled force probes

ii. Thermal periodontal probes• Advanced diagnostic aids in periodontal radiography

i. Digital radiography

ii. Substraction radiography

iii. Digital substraction radiography

iv. Transmission radiography

v. Magnetic resonance imaging

vi. Computerized tomography

vii. Nuclear medicine bone scan

• Advanced diagnostic aids in microbiologic analysis

i. Advances in culturing technique

ii. Advances in immunodiagnostic methods

iii. Advances in enzymatic methods

iv. Advances in nuclear biology- PCR and DNA probes

• Advanced diagnostic aids in charting the host response

i. Assessment of inflammatory mediators and products

ii. Assessment of tissue breakdown products

iii. Assessment of host derived enzymes

• Advanced diagnostic aids to determine periodontal disease activity

i. Crevicular contents• Products of bacteria• Products of host cells and host immunity

ii. Markers in peripheral blood• Neutrophil functional profile• Monocyte responsiveness to LPS• Circulating antibodies to plaque bacteria

iii. Detection of specific periodontal pocket bacteria

a. DNA probes

b. BANA hydrolysis

c. Antibody techniques

Advances in clinical diagnosis

• Degree of gingival inflammation

a. redness and swelling

b. gingival bleeding• Gingival bleeding ∞ plaque accumulation

gingival inflammation

(Greenstein G et al 1981)

size of inflammatory infiltrate

probability of losing attatchments

(Lang et al.1991)

Gingival temperature

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

(Kung et al 1990)• Commercially available system periotemp probe• 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

• Elevated subgingival temperature ∞ attatchment loss

& elevated propertions of

periodontopathic bacteria

( Haffajee et al.)

Periodontal probes

• Most widely used• Clinical assessment of connective tissue destruction in

periodontitis• Gold standard – recording changes in periodontal status• Probing depth is measured from the free gingival margin

(FGM) to the depth of the probable crevice.• not the most objective measure of loss of periodontal tissues

• CAL is a more objective measure of loss of Existing periodontal support.

• CAL also does not give any indication of current disease activity.

• When interpreting the PD and CAL measurements made with conventional periodontal probes, it is important to consider that these values depend on the inflammatory state of the tissues.

Classification of periodontal probes depending on generation.1.First generation probes:(conventional probes)Conventional manual probes that do not control 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

2.Second generation probe:

(Constant force probe)

Introduction of constant force or pressure sensitive probes allowed for improved standardization of probing.

e.g.: Pressure sensitive probe

Constant pressure probe

3.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 and three dimensional pocket that is being examined.

5.Fifth generation probe:(Noninvasive)

Three dimensional probe.

Basically these will add an ultrasound to a fourth generation probes.

If the fourth generation can be made, it will aim in

addition to identify the attachment level without

penetrating it.

e.g.: Ultra sonographic probe.

Florida probe:

Tip is 0.4mmSleeve- edge provides reference

to make measurementsCoil Spring; provides constant probing forceComputer for data storage.

• Disadvantages of Florida probe.Lack of tactile sensitivityFixed probing forceUnderestimation of deep periodontal pockets.Other electronic probes:Improvised Florida PASHA probeInterprobePerioprobeFoster Miller probeToronto Automated ( difficult to reproduce patient head

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

PSR system

• To screen dental patients to facilitate the detection of mild forms of periodontal diseases and to identify individuals who have previously undetected periodontitis.

• It is designed for general dental practitioner to identify the patient’s requiring periodontal care and to determine the type of care required.

• Based on the worst site per sextant.• If one or more sextants show significant signs of disease,

clinician is advised to do a complete periodontal examination and charting

Advances in Radiographic Assessment

• Dental Radiography are traditional method to assess destruction of alveolar bone.

• 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.

Digital Radiography

• Computerized images.• Image property almost equal to conventional radiographs• 1/3rd to half reductions in radiation dose.

Substraction Radiography

• Well established in medicine• Introduced in Periodontal diagnosis• Principle: Serial radiographs converted to digital images

superimposed composite image– Quantitative changes• Changes in density and volume of bone

a. can be detected as lighter areas (bone gain)

b. Dark areas (bone loss)

• Perfect accuracy at a lesion depth of 0.49 mm

( Grondhal et al 1988)

• Limitations: needs paralleling technique and accurate

superimposition.

Diagnostic Subtraction Radiography

• Positioning device during film exposure

specialized software designed for digital image subtraction

using conventional personal computers

• Applies an algorithm that corrects for the effects of angular alignment discrepencies and provides some degree of flexibility in imaging procedure

Computer Assisted Densitometric Image Analysis(CADIA)

Video cameras

measures

light transmitted through Radiographs

signals from camera

converted into

gray scale images

camera interfaced with image processor

+ computer

• Offers objective method for following alveolar bone density changes quantitatively over time.

• Higher sensitivity• High degree of reproducibility• accuracy

COMPUTED TOMOGRAPHY

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

• 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 axial slices are obtained by several revolution of the X-ray beam until the whole area of interest is covered.

• With the help of a computer and sophisticated Algorithms these slices are then used to generate a three dimensional digital map of the jaw 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.

ADVANTAGES of Computed Tomography

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 Tomography 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.

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

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

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

• The 3- D CBCT images canbe combined with high precision 3D visible light (photographic) surface images to create a virtual patient that accurately displays both hard and soft tissue structures.

• This multi- modal image image visualization enables a treatment platform that allows assessment of the patient’s present condition, planning and stimulation of treatment options, progress monitoring and evaluation of outcomes.

Advances in Microbiologic Analysis

• 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

• Microbiologic tests( that can identify)

a. Can support diagnosis of various Periodontal disease.

b. Can tell about initiation & progression

c. Active & Inactive

d. Can also be used to monitor Periodontal therapy

whether it is suppressed/ eradicated. • Several studies

(-)nce of Pathogens better periodontal health

(+)nce of pathogens periodontal disease

• Bacterial culturing• Direct microscopy• Immunodiagnostic methods• Enzymatic methods• Diagnostic assays based on molecular biologic techniques

Bacterial culturing

• Historically, widely used in characterizing composition of subgingival microflora.

• Plaque sample anaerobic culture

selective non-selective

Advantage: 1. clinician can obtain absolute or relative count

2. invitro method for antibiotic susceptibility

• Limitations :

i. Culture can grow only live bacteria.

ii. Sensitivity is low

iii. Requires sophisticated equipment and experienced personnel.

iv. Time consuming

v. expensive

Direct microscopy

• Dark field

Direct microscopy alternative to culture methods

• Ability to assess morphology

& motility in plaque.• Dark field microscopy seems an unlikely candidate as a

diagnostic test of destructive periodontal diseases.

Immunodiagnostic methods

• It employs Antibody

that recognize

specific bacterial antigen• Various procedures:

a. Direct & indirect immunofluorescence assays

b. Flow cytometry

c. ELISA

d. Membrane assay

e. Latex agglutination

Immunofluorescence assay

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

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

Direct IFAIndirect IFA

Cytofluorography/ Flow cytometry

Bacterial cells+ species specific AB

+ Secondary FL Conjugated AB Introduced in flowcytometer

Bacterial cells is separated into

single cell suspension-

passes through the tube

Cells identified by lasers

• merits :

rapid identification

labels bacterial cells species specific antibody

fluorescin conjugated secondary

antibody• Demerits:

sophistication

expensive

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)

• Latex Agglutination Test

Latex 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.

Advantages:Simple and Rapid testing

Higher sensitivity and specificity.

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.

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

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.

Molecular Biology Techniques

• 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 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

Whole 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.

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:6. Expensive 7. Expert personnel to carry out the test8. 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:4. Small quantity needed for reaction may not contain the

necessary target DNA5. Plaque may contain enzymes which may inhibit these

reactions.

Advances in characterizing the host response

• Diagnostic tests have been developed that add measures of the inflammatory process to conventional clinical measures.

• These diagnostic tests gives information about

pattern of destruction

current activity of disease

rate of disease progression

extent & severity of further breakdown

response to therapy

• So, knowing about disease destructive process-

clinician can individualise their therapeutic approach,

and customize the treatment.• Source of samples may be; GCF, Saliva, or Blood.• GCF is most commonly used, where as saliva is recently been

researched.• Assessment of Host response

i. Inflammatory mediators and products

ii. Host derived enzymes

iii. Tissue breakdown products

Inflammatory mediators & products:

• Cytokines- potent local mediators of inflammation

produced by variety of cells• GCF- TNF α

IL-1

IL-6

IL-8• Good correlation with disease status and severity but not

disease progression

• In untreated periodontitis- concentration of PGE2 increased during active phase of periodontal destruction.

• Cytokines ∞ disease severity & status• PGE2 ∞ active phase of periodontal disease

Host derived enzymes:

• Matrix components are dissolved by either

a. ECM metalloproteinase dependent

b. Plasmin dependent cleavage reactions

• Proteases and enzymes involved in these processes may be used as diagnostic aids.

• Aspartate aminotransferases• Collagenase• β-glucuronidase• Lactate dehydrogenase• Arylsulfatase• Elastase• Alkaline phosphatase

Tissue Breakdown Products

• Hydroxyproline• Glycosaminoglycans

For periodontal diagnosis, the ideal diagnostic test should be • Quantitative.• Highly sensitive method capable of analyzing a single

periodontal site in health as well as disease.• Reproducible.• Highly specific.• Simple to perform.• A rapid, one or two stage procedure.• Non-invasive.• Versatile in terms of sample handling, storage and transport.• Amendable to chairside use.• Economical. ( Chapple 1997)

Chairside periodontal test kits can be categorized as• Microbiologic tests• Biochemical test kits• Genetic kits

Microbiological test kits

The bacteriological tests

(Microscopy, Culture, Omnigene, Affirm DP and Evalusite) are mainly aimed at spirochetes, Aa, Pg and Pi.

Microbial tests can also be used to monitor periodontal therapy directed towards the suppression or eradication of periodontopathogenic  microorganisms

Omnigene

• DNA probe systems

• available for the detection of A. actinomycetemcomitans, P. gingivalis, P. intermedia, F. nucleatum, C. rectus, T. denticola

and E. corrodens.

Evalusite

• Evalusite is a kit that employs a novel membrane-based enzyme immunoassay for the detection of three putative periodontopathogens: Aa, Pg and Pi.

•  a pink spot is displayed if the test organism is present.• The main weaknesses of this test kit reside in• 1) the assumption that the three detected organisms are

causing disease;• (2) it is a multistage test; • (3) it has a subjective calorimetric end point and (4) there is no

permanent record of the results.

PerioScan®

• Perioscan is a diagnostic test kit that utilizes the BANA (N-benzoyl-DL-arginine-2-naphthylamide)-hydrolysis reaction,

• developed to detect bacterial trypsin-like proteases in the dental plaque

Biochemical test kits

• Perio 2000- to evaluate VSCs• Prognos- Stik- to detect elevated levels of MMPs• Perio- Check- to measure neutral protease activity• PerioGard- to detect AST• Pocket Watch- simple method for AST

Genetic test kits

• Various gene polymorphisms are considered to be risk factors for the initiation or progression of periodontal disease.

• In 1997, Kornman et al. found an association between the polymorphism in the genes encoding for interleukin-1α and interleukin-1β and increased severity of periodontitis.

• PST® genetic susceptibility test - the first and only genetic test that analyzes two interleukins (IL-1α and IL-1β) genes for variations. IL-1 genetic susceptibility may not initiate or cause the disease but rather may lead to earlier or more severe disease

Salivary diagnostics

 • OralDNA® Labs has developed a salivary test, the

MyPerioID® PST®, that identifies a patient’s genetic susceptibility and inherent risk to periodontal disease by evaluating their interleukin-1 (IL-1) gene cluster,

• MyPerioPath®, that identifies the type and concentration of 13 pathogenic bacteria known to cause periodontal disease.

Conclusion

• In periodontology, the success of any treatment is dependent upon the accuracy of the initial diagnosis.

• At present, the majority of chronic periodontitis cases can be adequately managed using existing diagnostic methodology, although it is clearly more desirable to be able to diagnose “active disease” as it occurs, rather than months later. 

•  However, the clinician must ensure that the use of such tests will benefit the patients in terms of both the value of diagnostic data obtained and the cost in time and money. 

References

• Carranza’s Clinical Periodontology 10th and 11th edition.• Armitage GC, Jeffcoat MK, Chadwick DE: longitudinal

evaluation of elastase as a marker for the progression of Periodontitis, J Periodontol 1994; 65:120.

• Beck JD: Issues in assessment of diagnostic tests and risk for Periodontal diseases, Periodontol 2000;7:100

Thank you