Advanced Diagnostic Aids

Dr. Khushbu mishra

HKEs 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
Genetic
response

systemic behavioural
 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 MichiganO 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 0.4mm
Sleeve- edge provides reference
to make measurements
Coil Spring; provides constant probing force
Computer for data storage.
 Disadvantages of Florida probe.
Lack of tactile sensitivity
Fixed probing force
Underestimation of deep periodontal pockets.
Other electronic probes:
Improvised Florida PASHA probe
Interprobe
Perioprobe
Foster Miller probe
Toronto 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
patients 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 patients
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
 Indirect IFA
Direct 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 its 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 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.
Disadvantage:
1. Expensive
2. Expert personnel to carry out the test
3. 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 conditions
3. Many species can be detected simultaneously
Disadvantage:
1. Small quantity needed for reaction may not contain the
necessary target DNA
2. 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
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
-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-1 and
interleukin-1 and increased severity of periodontitis.
PST genetic susceptibility test - the first and only genetic
test that analyzes two interleukins (IL-1 and IL-1) 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 patients 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
Carranzas 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