ADVANCED

DIAGNOSTIC AIDS

CONTENTS
INTRODUCTION
ADVANCES IN CLINICAL DIAGNOSIS
ADVANCES IN RADIOGRAPHIC MEASUREMENT
ADVANCES IN MICROBIOLOGIC ANALYSIS
ADVANCES IN CHARACTERIZING HOST
RESPONSE
ADVANCES IN GENETIC ASSESSMENT
ADVANCED DIAGNOSTIC AIDS IN DETECTING
HALITOSIS
ADVANCED DIAGNOSTIC TOOL TO STUDY
OCCLUSAL STRESSES
CONCLUSION

 Introduction
Periodontal diseases - prevalent human diseases defined by the signs and symptoms of
gingival inflammation and/or periodontal tissue destruction. Clinical diagnosis of
periodontitis - measuring the loss of connective tissue attachment to the root surface
(clinical attachment loss) and loss of alveolar bone (radiographic bone loss) .
Provides evidence of past periodontal destruction, its extent and severity.
Does not provide any information on the cause of the condition, on the patient's
susceptibility to disease, whether the disease is progressing, whether it is in
remission or whether the response to therapy will be positive or negative.
Disease process itself is considered to be site specific and has a multifactorial origin
Consideration should be given to including microbiologic, immunologic, systemic,
genetic, and behavioral factors, in addition to the traditional clinical and
radiographic parameters, when assessing patient status.

ADVANCES IN CLINICAL DIAGNOSIS

Gingival bleeding
Clinical evaluation - degree of gingival inflammation - redness and swelling of the
gingiva + gingival bleeding.
Gingival bleeding is a sensitive clinical indicator of early gingival inflammation.
Clinical advantage of being more objective.
Good indicator of the presence of an inflammatory lesion in the connective tissue
Severity of bleeding increases increase in size of the inflammatory infiltrate.
Evaluated- periodontal probe or a wooden interdental cleaner
.

Gingival bleeding universally considered indicator of gingival inflammation

and, by some indicator of disease activity.
Relationship to disease progression- unclear
Lang et al - retrospective study, reported that sites that bled on probing at
several visits had a higher probability of losing attachment than those that
bled at one visit or did not bleed. well controlled longitudinal studies
investigated the predictive values of such clinical signs, trying to correlate
them with attachment loss, but failed to demonstrate a significant correlation
between bleeding on probing and other clinical signs and subsequent loss of
attachment.
Limitation - healthy sites may bleed on probing. any force greater than 0.25
N evoke bleeding

Gingival Temperature :
Kung et al - thermal probes are sensitive diagnostic devices early inflammatory changes in the gingival tissues.
Studies - suspected active periodontitis lesions can create
measurable elevations in sulcular temperature.
PerioTemp probe (Abiodent, Inc., Danvers, Mass) - pocket
temperature differences of 0.10 C.
A naturally- temperature gradient - between maxillary and
mandibular teeth and between posterior and anterior teeth.

Individual temperature differences are compared with those

expected for each tooth.
Higher temperature pockets are signalled with a red emitting
diode.
Haffajee et al used this probe to assess its predictability in
identifying loss of attachment, concluding that sites with a red
(higher) temperature indication had more than twice the risk for
future attachment loss than did those with a green indication.
However, the influence of pocket depth on temperature is still not
clear, and further studies are needed to demonstrate the accuracy
of this device and its utility in clinical diagnosis.

Periodontal Probing
Classification
a) first generation probes/ conventional
b) second generation probes/pressure sensitive
c) third generation probes/ computerized

Most widely used diagnostic tool - clinical assessment of connective

tissue destruction in periodontitis .
Increased probing depth and loss of clinical attachment are pathognomic
for periodontitis..
Use of the periodontal probe - many problems in terms of sensitivity and
reproducibility of the measurements.
Clinical pocket depth obtained - not normally coincide with the
histologic pocket depth.
Tissue inflamed- offers less resistance to probe penetration, and the
probe tip either coincides with or is apical to the coronal level of
connective tissue attachment.
Healed gingiva - subgingival instrumentation -increased resistance to
periodontal probing.

Disparity - measurements depends on the probing technique,

probing force, size of the probe, angle of insertion of the probe,
and precision of the probe calibration.
All of these variables contribute to the large standard deviations
(0.5 to 1.3 mm) in clinical probing results, which make detection
of small changes difficult.
Mid 1980s, different probe prototypes -developed and tested to
overcome these limitations.
One of the main problems in reproducibility - variation in probing
force.
Penetration of the probe - positively correlated with probing force.

Solved - development of pressure

sensitive probe.
Studies have shown that with
forces of up to 30 g, the tip of the
probe seems to remain within the
junctional epithelium, and forces of
up to 50 g are necessary to
diagnose periodontal osseous
defects.
Standardization of probe tips (less
than 1 mm) and use of registration
stents - reproducible probing
angulation - used to overcome
sources of error.
Fabrication of stents - time
consuming and impractical for
clinical diagnosis.

Gibbs et al - developed Florida Probe System (Florida

Probe Corporation)
Probe tip - 0.4 mm in diameter. This probe tip
reciprocates through a sleeve, and the edge of the
sleeve provides a reference by which measurements
are made.
These measurements are made electronically and
transferred automatically to the computer when the
foot switch is pressed.
Constant probing force is provided by coil spring inside
the probe hand piece and digital readout.
Advantages
constant probing force
precise electronic measurement
computer storage of data
Dis-adv
Lack tactile sensitivity - independent movement,
which forces the operator to predetermine an
insertion point and angle.
Use of a fixed force setting throughout the mouth,
regardless of the site or inflammatory status, may
generate inaccurate measurements or patient
discomfort.
One common problem reported in different studies
where the Florida Probe System has been compared
with conventional probing is the underestimation of
deep probing depths by the automated probe.

In fact, several studies have suggested that the use of this

automated probing system does no offer any advantage
over conventional probing, rendering a similar level of
reproducibility.
However, other studies have clearly shown that with the
use of trained operators and performing the double
pass method the measurements taken with the Florida
Probe System are significantly less variable (lower
standard deviation) than those obtained with a
conventional probe.

Magnusson et al obtained mean standard deviations (reproducibility) for clinical

attachment level measurements of about 0.3 mm, which is clearly superior to an
average of 0.82 mm, with a range of 0.52 to 1.30 mm reported by Haffajee et al
using manual probing.
An electronic probe using an optical encoder transduction element - evaluated by
Goodson and Kondon.
These authors reported somewhat higher reproducibility with the electronic probe
compared to conventional probing and a correlation coefficient of 0.82 between
the two different methods.
This probe has now been named interprobe (Bausch and Lomb).
They provide constant probing force, computer storage of data, and precise electronic
management of the resulting inflammation.
Clinical evaluations of these systems- reported slightly improved probing, although
not clinically significant

The electronic probe described - Jeffcoat et al (the Foster Miller probe)

Capable of coupling pocket depth measurement with the detection of the cemento
enamel junction from which the clinical attachment level is automatically detected.
Never - released for general use.
Researchers at the University of Toronto have also described a probe (the Toronto
Automated probe) that, like the Florida probe, uses the occlusal incisal surface to
measure relative clinical attachment levels.
The sulcus is probed with a 0.5 mm nickel titanium wire - extended under air
pressure.
Controls angular discrepancies - mercury tilt sensor that limits angulation within
30 degrees, but it requires reproducible positioning of the patients head and
cannot easily measure second or third molars.
Two other electronic probes (the Birek Probe and the Florida Disk Probe) have been
designed to measure changes in attachment level using the occlusal surface or the
incisal edge as a reference point.
The Birek Probe works on constant air pressure and measures attachment level from
the occlusal surface.
In a study of duplicate measurements in nine subjects, it was found that 82% of the
measurements were within a 1 mm difference.

Low study compared- two models of the

Florida Probe ; the stent model and
the new disk model. The two probes
are similar in design (both have a
probe tip diameter of 0.4 mm and are
preset at a constant 25-g force) but
differ in the way they rest on a fixed
reference point. The probe tip of the
stent model runs through a metal
sleeve which has a 1 mm collar that
rests on the ledge of a fabricated
vacuform stent. The disk model has a
metal disk 11 mm in diameter that
rests on the occulusal surface or
incisal edge of the tooth. Both probes
are connected to a digital readout.
When the probe tip is in correct
alignment with the tooth and the
collar or disk is placed on the
respective fixed reference point, a
foot activated rheostat is depressed
to enter the measurement (to 0.1
mm) automatically into a computer.

The new electronic probes appear to be superior to manual probes.

In the studies described the range of overall standard deviations for
repeated measurements of individual sites in different subjects
was 0.17 to 0.32 mm. Regarding the ability to detect significant
attachment level changes, this should still be an improvement
over the 0.82 mm obtained by Haffajee et al using the manual
probe.

Probes and the examination of

patients considered for implants
Examination of patients - considered
for implants includes both clinical
evaluation of soft tissues and a
radiographic evaluation..
Probing around implants is difficult
(1) the prosthetic construction may
need to be removed for access
(2) standard metal instruments are
unsuitable.
Instead, plastic or titanium probe tips
should be used to avoid damage of
the implant / tissue interface. If
automatic probing is considered,
the Florida Probe is available with
a titanium tip that will not hurt the
implant ; also, the Interprobe
system comes with disposable
plastic tips.

ADVANCES IN RADIOGRAPHIC MEASUREMENT

Radiographs cannot accurately reflect - bone morphology buccally and lingually
provide -useful information on interproximal bone levels.
Provide information on the periodontium that cannot be obtained by any other
noninvasive methods (e.g., root length, root proximity and presence of periapical
lesions and estimates of remaining alveolar bone).
Substantial volumes of alveolar bone must be destroyed before the loss is detectable in
radiographs, specifically, more than 30% of the bone mass at the alveolar crest must
be lost for a change in bone height to be recognized on radiographs.
Therefore conventional radiographs are very specific, but lack sensitivity.
This low degree of sensitivity - due to the subjectivity of radiographic assessment and
to the inherent sources of variability affecting the conventional radiographic
technique- (1) variations in projection geometry ; (2) variations in contrast and
density due to differences in film processing, voltage and exposure time ; and (3)
masking of osseous changes by other anatomic structures.

The variations in projection geometry - reduced by the use of well standardized

long cone parallel radiographic techniques.
To standardize the radiographic assessment, radiographs should be obtained in a
constant and reproducible plane, using film holders with a template containing
some kind of impression material, which is placed in a constant position on a
group of teeth, and an extension arm that can be precisely attached to both the
film holder and the x ray tube.
The use of a parallel radiographic technique should be standard to all
radiographic assessments for periodontal diagnosis. The use of individualized
film holders has been shown to be valid in evaluating bone changes in
longitudinal studies and clinical trials.

Digital radiography :
Variations in image quality - variables inherent to conventional radiography reduced with the use of digital intraoral radiography.
Digital radiography enables the use of computerized images - stored,
manipulated, and corrected for under and overexposures.
Yield almost equal image properties compared with conventional radiographs,
but through digital storage and processing, diagnostic information can be
enhanced.
Dose reduction obtained with this technique (between 1/3 to of dose reduction
compared with conventional radiographs).
Two digital radiography systems rely on the sensor - the direct and indirect
methods The direct method uses a change coupled device (CCD) sensor
linked with a fiber optic or other wire to the computer system.
This direct digital radiography - real time imaging, offering both the clinician
and the patient an improved visualization of the periodontium by image
manipulation and comparison with previously stored images.

Disadvantage - limited sensor area, which is only large enough to depict one or two teeth.
The sensor rigidity attached to a wire- sterility issues- ideal image projection by using film
holders very difficult.
The indirect method (Digora System) - phosphor luminescence plate, which is a flexible film
like radiation energy sensor placed intraorally and exposed to conventional x ray tubes. A
laser scanner reads the exposed plates offline and reveals digital image data, which can be
enhanced, stored, and compared with previous images.
Advantage of this indirect method is due to the plate size and flexibility - almost identical to
conventional x ray films - paralleling technique with the use of film holders can be easily
applied.
Digital intraoral radiography is in a state of rapid development. Sensors, as well as computer
hardware and software, are continually modified and improved.
Due to the clear advantage of real or almost real images that can be improved and to the
important educational component of online images presented to the patient, it is expected that
digital radiography will soon replace conventional radiography in modern daily practice.
However, certain improvements should be expected in order to overcome some of the current
limitations.

Detecting Osseous Changes Between Radiographic Examinations :

All radiographic methods simply image the existing anatomy of the teeth and
supporting tissue- do not indicate the rate or presence of active bone or
attachment loss, nor do they indicate whether past episodes of destruction and
healing have occurred. To do so, the clinician must compare two or more
carefully exposed radiographs taken at different examinations.
Most assessment of bone loss in clinical practice today is achieved by visual
comparison of the radiographs. The radiograph contains so much information
that it is difficult for the human eye to detect small changes in bone support in
the presence of a busy background containing the teeth and cortical and
trabecular bone. Studies have shown that a 30% to 50% change in bone
mineral is needed to be visible even to the experienced clinician using
interpretative radiography.
Several methods have been developed to allow measurement of bone height on
serial radiographs. The simplest method involves measuring the distance from
the CEJ to the alveolar crest. Of course, this method is limited both by the
geometry of the film and the ability of the clinician to detect the alveolar crest.
Methods have been developed that, in part, correct for errors in angulation.
Bone loss may be expressed as a percentage of the root length to partially
correct for errors due to foreshortening or elongation. This method, like the
direct measurement method, is also limited by the ability of the clinical to
accurately detect the alveolar crest. Nonetheless, such methods have
substantial sensitivity in experienced hands. Changes in bone height as small
as 0.1 mm may be detected.

Subtraction radiography :
A well established technique in medicine - introduced as a technique in periodontal
diagnosis.
Shown (1) a high degree of correlation between changes in alveolar bone determined
by subtraction radiography and attachment level changes in periodontal patients
after therapy and (2) increased detectability of small osseous lesions compared with
the conventional radiographs from which the subtraction images are produced.
Grondahl et al, using subtraction analysis - perfect accuracy at a lesion depth
corresponding to 0.49 mm of compact bone - three times larger to be detectable with
a conventional radiology technique.
Shown a degree of sensitivity similar to that for I 125 absorptiometry - detect a change in
bone mass of as little as 5%.
Subtraction radiography has been applied to longitudinal clinical studies. Hausmann et
al detected significant differences in crestal bone height of 0.87 mm, and Jeffcoat et
al showed a strong relationship between probing attachment loss detected using
sequential measurements made with an automated periodontal probe and bone loss
detected with digital subtraction radiography.

Disadvantage : need to be close to identical projection alignment

during the exposure of the sequential radiographs, which makes
this method very impractical in a clinical setting.
Recently, new image subtraction methods (diagnostic subtraction
radiography (DSR) have been introduced combining the use of
a positioning device during film exposure with specialized
software designed for digital image subtraction using
conventional personal computers in dental offices.
This image analysis software system applies an algorithm that
corrects for the effects of angular alignment discrepancies and
provides some degree of flexibility in the imaging procedure.
Recently, the use of the DSR technique has been compared with the
conventional subtraction radiography technique yielding
statistically significant gains in diagnostic accuracy over
conventional radiographs and no differences against the classical
subtraction radiography technique

Use of subtraction radiography or CADIA presupposes that the radiographs are taken
with similar contrast, density, and angulation.
Color coding the subtraction images improves the ability of the novice clinician to
detect bone loss or gain. In the color images, bone gain - green and bone loss -as
shades of red.
Computed Tomography :
Specialized radiographic technique that allows visualization of planes or slices of
interest.
Unlike conventional tomography, which blurs all structures not in the plane of interest,
computed tomography actually removes the structures not in the plane of interest,
resulting in an image with a clear visualization of the slice through the organ (s)
under study.
To accomplish this, multiple exposures are taken using known geometry around the
area of interest. Specialized hardware, known as the CT scanner, is used to
accomplish this. Software is then used to reconstruct each plane or slice of interest.
Since CT equipment is expensive and the procedure carries a relatively high radiation
burden for the patient - use should be reserved for questions that cannot be
answered via clinical examination and / or conventional transmission radiography.

Magnetic resonance imaging

Fundamentally different from the other imaging techniques and does not use ionizing
radiation.
Unlike conventional radiographic techniques, the hard tissues, bone, and teeth are not
strongly imaged
The strength - ability to image soft tissue.
To acquire an MRI image the patient is placed in a strong magnetic field. The protons
of the hydrogen nuclei of the water within the tissues precess (rotate like a
spinning top) about the direction of the magnetic field. Resonance frequency
energy is applied and then removed. The response of the nuclei to the resonance
frequency stimulation is observed in a receiver coil.
Based on these principles it is not surprising to find that alveolar bone is only weakly
imaged, whereas soft tissue, such as salivary glands, dental pulp, and the disc of
the temporomandibular joint, may be easily observed if the correct scanning
parameters are used.
.

Advantages - noninvasive and we are presently unaware of

significant biological risks.
Dis-advantages: expensive, requires considerable scan time for high
resolution images, and may be claustrophobic for the patient.
MRI is prone to some of the same errors as CT, including motion
and volume averaging artifacts.
MRI is a rapidly evolving field and its usefulness to dental
applications is under investigation

Nuclear Medicine Bone Scans

Branch of radiology that uses radiolabeled pharmaceuticals that are specifically
intended to image particular organs or detect specific disease processes.
For the diagnosis of periodontal disease, bone scans have been used to detect sites of
active bone loss. The radiopharmaceutical is a technetium labeled
disphosphonate called 99m Tc methylene diphosphonate.
The diphosphonate moiety is the bone seeker that is adsorbed onto the forming front
of bone that occurs either during bone apposition or behind the bone resorption.
The radiolabel, technetium, is a synthetic element with a 6 hour physical half life.
The biological half life of the radiopharmaceutical is 3 hours.
The bone scan does not image the anatomy of the area of interest. Rather, it detects
alteration in bony metabolism that may occur prior to radiographically detectable
changes.
It is also important to note that these changes are not specific to a particular disease.
therefore, the bone scan must be used in conjunction with clinical findings and
radiographs in order to make an accurate diagnosis.

To perform a bone scan, the radiopharmaceutical is injected intravenously. Following

a period to allow for bony uptake of the agent, uptake is either imaged using a
gamma camera or measured using specially designed detectors for intraoral use.
Areas of active bone loss appear as hot spots in the image.
Studies have shown that the sensitivity and specificity of this technique in
prognosticating alveolar bone loss due to periodontitis subsequently determined
radiographically is over 90%.
Bone scans are best used to determine whether a patient has active sites of bone loss
and could benefit from an experimental treatment, or to determine whether a
patient who is to undergo a bone marrow transplant has sites of active periodontal
disease or occult disease that need immediate attention.

Advances in Microbiologic Analysis :

Subgingival oral bacteria - main initiating agents in the development of
periodontal disease.
These microbiologic tests - potential to support the diagnosis of the various forms
of periodontal disease, to serve as indicators of disease initiation and
progression (i.e., disease activity), and to determine which periodontal sites
are at higher risk for active destruction.
Microbial tests can also be used to monitor periodontal therapy directed at the
suppression or eradication of periodontopathic microorganisms.
Several methods have been employed for the detection of putative periodontal
pathogens in subgingival samples. Some of these methods have been strictly
used for research purposes, whereas others have been adapted or modified for
clinical use.

All of these methods share the common need for an appropriate

subgingival plaque sample.
Selecting the proper specimen site and collecting an adequate
sample are essential elements in periodontal microbiology.
These samples may be difficult to obtain in patients infected by
organisms that are unevenly distributed in the dentition.

Bacterial Culturing :
Considered the reference method
(gold
standard)
when
determining the performance of
new
microbial
diagnostic
methods.
Plaque samples are cultivated
anaerobically and by using
selective and nonselective media,
together with several biochemical
and physical tests, the different
putative pathogens can be
identified.
Advantage :Obtain relative and
absolute counts of the cultured
species.
Only in vitro method - assess for
antibiotic susceptibility of the
microbes

Shortcomings: Only grow live bacteria, therefore strict sampling and transport
conditions are essential.
Putative pathogens, such as Treponemas sp. and Bacterioides forsythus are
fastidious and difficult to culture.
Sensitivity of culture methods is rather low, since the detection limits for selective
and nonselective media average 103 to 104 bacteria and hence low numbers of
a specific pathogen in a pocket - undetected.
Important drawback is that culture requires sophisticated equipment and
experienced personnel and is relatively time consuming and expensive.
Clinicians, when using this methods, must be confident that the laboratory has the
appropriate technology and expertise in periodontal microbiology to
communicate diagnostically and therapeutically useful information to them.

Direct microscopy
Darkfield or phase contrast microscopy - suggested as an alternative to culture
methods - ability to directly and rapidly assess the morphology and motility of
bacteria in plaque sample.
Used to indicate periodontal disease status and to structure maintenance
programs.
However, most of the main putative periodontopathogens, including
Actinobacillus actinomycetemcomitans, P. gingivalis, B, forsythus, Eikenella
corrodens, and Eubacterium species, are non motile, and therefore this
technique is unable to identify these species.
It is also unable to differentiate among the various species of Treponema.
Seems an unlikely candidate as a diagnostic test of destructive periodontal
diseases

Immunodiagnostic Methods
Immunological assays employ antibodies that recognize specific bacterial
antigens to detect target microorganisms.
Revealed using a variety of procedures:
Direct and indirect immunofluorescent microscopy assays (IFA)
Flow cytometry
Enzyme linked immunoabsorbent assay (ELISA)
Membrane assay
Latex agglutination.
Direct IFA - monoclonal and polyclonal antibodies conjugated to a fluorescein
marker that binds with the bacterial antigen to form a fluorescent immune
complex detectable under a microscope.
Indirect IFA employs a secondary fluorescein conjugated antibody that reacts
with the primary antigen antibody complex.
Both direct and indirect immunofluorescence assays are able to identify the
pathogen and quantify the percentage of the pathogen directly using a plaque
smear.
IFA has been used mainly to detect A. actinomycetemocomitans and P. gingivalis.

Zambon et al - technique is comparable to bacterial culture in its ability to

identify these pathogens in subgingival dental plaque samples.
In fact immune fluorescence microscopy may be even more likely to detect
them in clinical samples because it does not require viable bacterial cells.
Comparative studies indicate that the sensitivity of these assays ranges from 82%
to 100% for detection of A. actinomycetemocomitans and from 91% to 100%
for detection of P. gingivalis, with specificity values of 88% to 92% and 87%
to 89%, respectively.

Cytofluorography or flow cytometry - rapid identification of oral bacteria

involves labeling bacterial cells from a patient plaque sample with both
species specific antibody and a second fluorescein conjugated antibody.
The suspension is then introduced into the flow cytometer, which separates
the bacterial cells into an almost single cell suspension by means of a
laminar flow through a narrow tube.
The sophistication and cost involved in this procedure precludes its wide usage.

ELISA

Similar in principle to other radioimmunoassays, but an enzymatically derived

color reaction is substituted as the label in place of the radioisotope.
The intensity of the color depends on the concentration of the antigen and is
usually read photometrically for optimal quantitation .
Used primarily to detect serum antibodies to periodontopathogens ; however it
has also been used in research studies to quantify specific pathogens in
subgingival samples using specific monoclonal anibodies.

Membrane immunoassay
Recently marketed (Evalusite).
It involves linkage between the antigen and a membrane bound antibody to
form an immunocomplex that is later revealed through a colorimetric reaction.
Evalusite - designed to detect A. actinomycetemcomitans, P. gingivalis, and P.
intermedia.
Immunological assays for oral bacteria, although extensively used for research
purposes, lack the clinical validation since most of them have never been
available commercially.
Cross reactivity leading to detection of false positives may represent a major
problem, mostly when polyclonal antibodies are used. On the other hand the
use of monoclonal antibodies may cuase the occurrence of false negatives
when compared with culture, due to their high specificity.

Immunological assays can identify dead target cells, thus not requiring stringent
sampling and transport methodology.
Cannot be used to determine antibiotic susceptibility.
Most of these assays provide a quantitative or semiquantitative estimate of target
microorganisms ; however, these methods generally show poorer detection
limits than nucleic acid probes of PCR assays.
Snyder et al tested the sensitivity of the Evalusite test, demonstrating a detection
limit of 105 for A. actinomycetemcomitans and 106 for P. gingivalis.

Latex agglutination
Very simple immunological assay based on the binding of protein to latex.
Latex beads are coated with the species specific antibody, and when these beads
come in contact with the microbial cell surface antigens or antigen extracts,
cross linking occurs ; its agglutination or clumping is then visible usually in
2 to 5 minutes.
Because of their simplicity and rapidity, these assays have great potential for
chariside detection of periodontal pathogens.
Two types of latex agglutination tests the indirect assay and inhibition assay.
The indirect assay is the most common latex agglutination test for bacteria.
The antibody is bound to latex. When a suspension of the plaque sample is mixed
with the sensitized latex and gently agitated for 3 to 5 minutes, resulting
agglutination or clumping is indicative of a positive result for the bacteria
being tested.
The inhibition assay is based on the principle of inhibiting the expected
agglutination reaction between known antigen and known antibody as a result
of competition.

Enzymatic Methods of Bacterial Identification

B. forsythus, P. gingivalis, the small spirochete Treponema denticola,
and Capnocytophaga species share a common enzymatic profile,
since all have in common a trypsin-like enzyme.
The activity of this enzyme can be measured with the hydrolysis of
the colorless substrate N benzoyl arginine 2 naphthylamide
(BANA). When the hydrolysis takes place, it releases the
chromophore - naphthylamide, which turns orange red when a
drop of fast garnet is added to the solution.
Diagnostic kits has been developed using this reaction for the
identification of this bacteria profile in plaque isolates
(Perioscan).
Loesche et al proposed the use of this BANA reaction in subgingival
plaque samples to detect the presence of any of these periodontal
pathogens and thus serve as a marker of disease activity. Using
probing depths as a measure of periodontal morbidity, Loesche et
al showed that shallow pockets exhibited only 10% positive
BANA reactions, whereas deep pockets (7 mm) exhibited 80% to
90% positive BANA reactions.

Beck et al used the BANA test as a risk indicator for

periodontal attachment loss. Taken collectively, results
using this diagnostic method suggest that positive
BANA findings are a good indication that T. denticola,
P. gingivalis, or both are present at sampled sites. One
of the potential difficulties of this test is that it may be
positive at clinically healthy sites and remains to be
proven whether this test can detect sites undergoing
periodontal destruction. Besides, since it only detects a
very limited number of pathogens, its negative result
does not rule out the presence of other important
periodontal pathogens.

BACTERIAL IDENTIFICATION BY
TOXINS & PROTEINS
TOPAS I Kit:
The TOPAS I (Toxicity prescreening
assay) is a chairside ; calorimetric
assay designed to detect 2 markers of
bacterial infection in gingival
crevicular fluid i.e. toxins and
proteins.
Rationale of TOPAS I
The approach used in TOPAS I is to
react the Toxin and protein samples
with a mixture of reagents to
produce a new set of compounds
which have one or more
chromophores. In the case of toxins
these chromophores all absorb
visible light in the yellow (420 nm)
region;whereas in the case of
proteins all these chromophores
absorb visible light in the blue (600
nm) region.

Toxin detection
The toxins are produced by pathogenic anaerobic bacteria isolated from active
periodontal disease sites which are measured at high (millimolar) levels in the
GCF.
TOPAS is calibrated using hydrogen sulfide as the toxic standard at levels ranging
from 0 mM (noassay) to 2mM (extreme toxicity). The toxins react with a
mixture of chemical reagents to produce yellow coloured by-products.
Toxins + Colourless reagent -> Yellow Colour
(bacterial)
Protein detection
Proteins include both bacterial proteins as well as human inflammatorv and serum
proteins such as antibodies and globulins. Bacterial infection increases the level
of these proteins in the GCF.
TOPAS is calibrated using Human serum albumin (HAS) as the protein standard
at levels ranging from none detectable (<5 ug) to abundant (>50 ug ). The
proteins react with the chemical reagents to produce blue colored by-products.
Proteins
+ Brown Dye -> Blue colour
(antibodies + Albumins + Enzymes)
The colour intensity of the toxins and protein solution is graded by result chart.

Deoxyribonucleic Acid Probe Technology

Nucleic Acid Probes. Entail segments of single
stranded nucleic acid, labeled with an
enzyme or radioisotope, that can locate and
bind to their complementary nucleic acid
sequences with low cross reactivity to
nontarget organisms.
Target whole genomic DNA or individual genes.
Whole genomic probes are more likely to cross
react with nontarget microorganisms due to
the presence of homologous sequences
between different bacterial species.
However, specific genes, such as 16S rRNA
(ribonucleic acid) genes, contain signature
sequences limited to organisms of the same
species.
These oligonucleotide probes display limited or
no cross reactivity with nontarget
microorganisms.

The assay can rapidly test for multiple bacteria, including

A.actinomycetemcomitans, P. gingivalis, B. intermedius, C. rectus,
E. corrodens, Fusobacterium nucleatum, and T. denticola in multiple
clinical plaque samples. The probes are able to detect as few as 10 2 to 104
bacteria, and the sensitivity and specificity are not affected by the
presence of unrelated bacterial in mixed culture samples.

Restriction Endonuclease Analysis

Recognize and cleave double stranded
DNA at specific base pair sequences.
The DNA fragments generated are separated
by electophoresis, stained with ethidium
bromide, and visualized with ultraviolet
light.
The genetic heterogeneity and homogeneity
of stains can then be evaluated by
comparing the number and size
(electrophoretic pattern) of the DNA
fragments obtained.
These DNA fragment patterns constitute a
specific fingerprint to characterize
each strain.
Powerful tool for determining the
distribution of a specific pathogenic
strain throughout a population.
This technology has also been applied to the
molecular genetic analysis of the natural
diversity of such oral bacteria as A.
actinomycetemcomitans, P. gingivalis, P.
intermedia, E. corrodens, F. nucleatum,
and T. denticola and has been very useful
in studying the transmission patterns of
putative periodontal pathogens among
family members.

Polymerase Chain Reaction

Amplification of a region of DNA flanked
by a selected primer specific for target
species.
The presence of specific amplification
product indicates the presence of the
target microorganisms. Among the
different nucleic acid assays.
Demonstrates the best detection limits, as
few as five to ten cells and shows no
cross reactivity under optimized
amplification conditions.
Different bacterial species may be detected
simultaneously by multiplex PCR in
which several distinct primer pairs,
each specific for a given target
microorganism are employed in a
single tube amplification process.
Drawback - relatively small aliquots are
used for the amplification process. If
this small quantity of the plaque sample
does not contain the targeted
microorganism, the assay will not
detect it.
Subgingival plaque may contain
enzymes that can alter the
amplification process.

Until recently, PCR assays were able just to assess the presence of the target
microorganism.
Fujise et al have developed a quantitative PCR method.
PCR assays have the potential for being an ideal detection method of periodontal
microorganisms.
It is relatively easy to perform and demonstrates excellent detection limits and
little cross reactivity under optimal conditions.

Advances In Characterizing The Host Response :

Diagnostic tests have been developed - measures of the inflammatory process to
conventional clinical measures.
Provide information on the destructive process itself, current activity of the
disease, rate of disease progression, patterns of destruction, extent and
severity of future breakdown, and likely response to therapy.
Clinician would be able to better individualize his therapeutic approach, thus
customizing the recommended treatment.
Assessment of the host response refers to the study of mediators, by immunologic
or biochemical methods, that are recognized as part of the individuals
response to the periodontal infection. These mediators are either specifically
identified with the infection, such as antibody to a putative pathogen, or
represent a less specific reaction like the local release of inflammatory
mediators, host derived enzymes, or tissue breakdown products.

Source of Samples:

GCF - more than 40 components of GCF - studied. Divided into three main
groups : host derived enzymes, tissue breakdown products, and
inflammatory mediators.

Gingival crevicular cells

Saliva - easily collected and may contain both locally and systemically
derived markers of periodontal disease.
No saliva based diagnostic tests are available to be used in clinical practice.
Proposed diagnostic markers in saliva include proteins and enzymes of host
origin, phenotypic markers, host cells, hormones (cortisol), bacteria and
bacterial products, volatile compounds and ions.

Blood serum

Blood cells

Urine

Immune response :

Antibody : total immunoglobulin and IgG subgroups -specific antibody or

total Ig in GCF appears to be of no use in distinguishing between stable and
progressive sites. Some evidence suggests that a reduction in specific
antibody in serum and consequently GCF in patients with existing disease can
place them at risk for further disease progression

Complement

Inflammatory response

Arachidonic acid derivatives, eg prostaglandin E2 (PGE2)

Cytokines, eg IL1, IL-2, IL-4, IL-6, TNF-.

IL 1 and are present in inflamed gingiva. They are also present in GCF
from patients with periodontitis and extremely low concentrations are found at
healthy sites. Their levels were reduced following scaling and root planing but
were not found to correlate with probing depth measurements.
TNF - is also present in GCF but does not correlate with probing depth or
gingival inflammation and its total amount was inversely related to tissue
inflammation
No significant differences in the mean level of IL 1 in refractory or stable
patients but refractory sites in this patients group produced significantly more
IL-6.

PGE2 levesl are

low - health
gingivitis - modest rise in GCF PGE2 - 32 and 53 ng/ ml
Untreated periodontitis patients - significantly higher levels than
gingivitis patients.
Levels greater than 66 ng/ml were found to be predictive of further
possible loss of attachment and this level was used as a cut of value in a
positive and negative screening test.
Diagnostic tests :
Although GCF PGE2 has considerable potential as a screening test for
periodontal activity strangely no commercial efforts are currently underway to
develop one.
In this regard, it is now possible to assay GCF PGE2 with an ELISA
assay using a monoclonal rabbit anit -PGE2 antibody.
Cytokines are also assayed using ELISA techniques which could be
developed into chairside kits.
However, at present the predictive ability of these markers is still in
doubt. Thus, the most likely diagnostic marker of the inflammatory and
immune factors described above is GCF PGE2.

Host Derived
Proteolytic
enzymes Hydrolytic enzymes
Enzymes
Aryl

sulphatase
Collagenase
- glucuronidase
Elastase
Alkaline
Cathepsin G
phosphatase
Cathepsin B
Acid phosphatase
Cathepsin D
Myeloperosidase
Dipeptidylpeptidases
Lysozyme
Tryptase
Lactoferrin.

COMMERCIAL DIAGNOSTIC KITS BASED ON GCF

PROTEOLYTIC AND HYDROLYTIC ENZYME LEVELS
Periocheck (ACTech) :
Detects the presence of neutral
proteinases such as collagenase in GCF.
A paper strip is used to obtain a GCF
sample. This strip is then placed in
contact with a collagen gel to which a
blue dye has been covalently bonded.
This is then incubated at 430 C. If the
neutral proteinases are present in the
sample they will attack the collagen gel
and release the blue dye.
The released blue dye produces a
blue colour in the strip, the intensity of
which is proportional to the amount of
enzyme present in the sample. The
intensity and the area of the colour is
then scored on a scale of 0 to 2 by
comparing it with three standards on a
colour card which is provided with the
test kit.

Prognostik (Dentsply)
This system detects the presence
of the serine proteinase, elastase, in
GCF samples.
A GCF sample is collected on
special paper strips which have been
impregnated with the appropriate
peptidyl derivative of 7 aminotrifluoromethyl coum arin (AFC).
The substrate used is MeOSuc AlaAla- ProVal AFC which detects
elastase and is linked to a fluorescent
leaving group, AFC.
If elastase is present the sample
reacts with the substrate in 4 8
minutes releasing the fluorescent
leaving group, AFC. This produces
green fluorescence in the strip which
can be seen under ultraviolet (UV) light
using a UV light box.
The intensity of the fluorescence
is proportional to the amount of GCF in
the sample and this is scored by
comparing it with AFC standards

Pocket Watch
CSA AST PP
inorganic
sulfite
Sulfite ion reacts
MGC- allowing pink
colored
rhabdomyine dye to
see through.

Advantages :

Some eg cathepsin B, elastase,

dipeptidyl peptidases II and IV, and
- glucuronidase, are predictive of
disease activity in longitudinal
studies.
Simple to use, particularly the
colour detection systems.
Can be read after a short time.
Can be shown to the patient and
related to the tooth site.

Disadvantages :

The choice of the most appropriate

biomarker may still be difficult at
the present state of knowledge.
There is difficulty in determining
the sites to sample and when to
sample them.
If a moiety is associated with
inflammation this may mask its
association with destructive
disease.
No account of biological control
mechanisms is taken in present
tests.
cost.

Periogard AST in GCF :

Test kit using paper point GCF samples and calorimetric detection.
The test kit consists of a tray with two test cells for each tooth and
appropriate reagents for conducting the test. The strip containing the GCF
sample is placed into suitable test well and two drops of one agent (10 mM Tris
HCL with 0.067% fiction X 100, pH 6.0) is added. At the same time positive
and negative control cells are prepared using strips provided. Two drops of a
solution provided (260 mMl - aspartic acid ; 33 mM 2 oxogluteric acid ; 4.3
mM disodium EDTA, 1.6% polyvinylpyrolidone, 0.067% Triton X- 100, 2.7 mm
sorbic acid in 100 MM Tris HCL, pH 6.0) are added to the wells and allowed to
incubate at room temperature.
After 9 minutes incubation the substrate / detection solution (1 mg fast
red RC diazotised salt in 1% methanol, 0.06% Triton X 100, in 230 Mm Tris
HCL, Ph 8.0) is mixed and two drops are added at 10 minutes. Five minutes later
the test results can be read by eye by comparing the test well colour to the colour
of the positve control.
A colour of greater intensity to that of the negative control is scored as
positive and one of lesser or equal intensity as a negative result. The test is
designed to be positive at 800 mIU AST activity and negative at values , 800
mIU

Markers of connective tissue degradation :

Soft tissue degradation products :
Fibronectin
Hydroxyproline containing
peptides
Glycosaminoglycans

Bone specific proteins:

Osteonectin and bone
phosphoprotein(N-Propeptide)
Osteocalcin
Cross linked carboxyterminal
telopeptide of type-I collagen

Methods of isolation and detection

The detection of osteonectin requires the use of
nitrocellulose strips as it cannot be recovered from conventional
strips. In contrast, GCF for the detection of osteocalcin and ICTP
was collected on conventional paper strips left in situ for 30
seconds. However, in some studies of osteocalcin and ICTP
multiple strip collection was used employing two in succession
for one minute at the same site for very short collection periods.
Clearly, some standardistion of collection technique is required if
the data from studies is to be compared.
The biochemical techniques used to isolate and detect
components in this section may be difficult to modify for chair
side use. However monoclonal and polyclonal antibodies have
been produced for osteonectin, ICTP and N- Propeptide. Using
these, osteocalcin has been assayed with either ELISA or radioimmunoassays, osteonectin and N-propeptide with ELISA and
ICTP with radio-imunoassays.

ADVANCES IN GENETIC
ASSESSMENT
The periodontitis susceptibility trait test is the
only genetic susceptibility test for severe
periodontitis that is commercially available.
It evaluates the simultaneous occurrence of allele
2 at the IL-1A +4845 and 1B +3954 loci. A
patient with allele 2 at both loci is considered
genotype positive and therefore more
susceptible to develop severe periodontitis.
Used to identify a predisposition to disease,
treatment approaches and outcomes will still
be influenced by environmental and behavioral
factors.
Gives no information on disease activity or
susceptibility

ADVANCED DIAGNOSTIC
AIDS IN DETECTING
HALITOSIS
GAS CHROMATOGRAPHY
The connection between bad breath
(halitosis) and certain volatile sulfur
compounds (VSC) was first
established by Tonzetich .
He determined that certain anaerobes
were the source of these compounds,
primarily methyl mercaptan,
hydrogen sulfide, and to a lesser
extent, dimethyl sulfide.
Oral chroma individually measures three
volatile sulfur compounds :hydrogen
sulfide, methyl mercaptan and
dimethylsulfide and correlates each
with cause.
Ideal as a pre-examination, preventive
dentistry device
.

Graphic displays are ideal for communicating with patients regarding their oral
health condition. Monitor patint improvement with historical displays.
The OralChroma including analysis software, computer connection cable, an
ample supply of plastic syringes for collecting breath samples

PROCEEDURE

HALIMETER
Gives a digital readout in parts per
billion (ppb) VSC, which is not
only quantitative, but is more
accurate than the very subjective
organoleptic method.
Using a recorder, in conjunction with
the Halimeter, offers a hard copy
record for both the dentist and
patient..
The only function of the Halimeter is
to serve as a reliable monitor for
the measurement of VSC
concentrations. If other chemical
agents cause bad breath, and might
exist in the breath sample, they may
or may not respond in the
Halimeter.

The Diamond Probe/Perio 2000

System is a dental device
designed to detect sulfide
concentrations of various forms in
gingival sulci.
The system combines a
conventional Michigan O style
dental probe with a sulfide sensor,
which measures PD, BOP, and
SUL, simultaneously
The micro-sulfide sensor responds
to sulfide ions and measures
metabolic-products of mainly
anaerobic bacteria and, indirectly,
bacterial activity.

Intended to be utilized as part of a total program encompassing a thorough

history and physical examination of the patient.
Along with an organoleptic assessment, the quantitative nature of Halimeter data
can serve as an excellent tool for following the progress of treating halitosis,
and for archiving hard copy records, halimeter data by itself cannot affirm
whether a breath problem exists - VSC levels can change with the time of day.
The dental practitioner is required to include the assessment of other diagnostic
procedures prior to making a positive conclusion.

ADVANCED DIAGNOSTIC TOOL TO STUDY

OCCLUSAL STRESSES
The finite element method is numerical method, which can be implemented to
solve many problems.
First developed in 1956 for the analysis of aircraft structural problems.
After within a decade, the potential of the method for the solution of different
types of applied sciences and engineering problems were recognized.
Over the years, the finite technique has been so well established that today it is
considered to be one of the best methods for solving a wide variety of
practical problems efficiently.

Application of fem
Finite element analysis as well as other related morphometric techniques such as
the macroelement and the boundary integral equation method (BIE) is useful
for the assessment of complex shape changes.
To the description of form changes in biological structures (morphometrics),
particularly in the area of growth and development.
For the understanding of stress related bone remodeling and also provides a
guideline reference for the design of dental implants.
Useful for structures with inherent material homogeneity and potentially
complicated shapes such as dental implants
Analysis of stresses produced in the periodontal ligament when subjected to
orthodontic forces
To optimize the design of dental restorations

Advantages

It is a non-invasive method

The actual stress at any point can be measured.

The actual displacement (initial tooth mobility) of the tooth can be measured.

The reproducibility does not effect the physical properties of the involved
material and the study can be repeated as many times as the operator wants.

CONCLUSION
Although there are many potential markers for periodontal
disease activity and progression, still neumerous features hamper
the ability to use them as diagnostic tests of proven utility. There
is still a lack of a proven gold standard of disease progression
and thus the correlation of these potential markers with proven
clinical attachment loss may be a potential confounder in any
proposed test.
After all these years of intensive research we still lack a
proven diagnostic test that has demonstrated high predictive
value for disease progression, has a proven impact on disease
incidence and prevalence and is simple, safe and cost effective.

REFERENCES:
o
o
o
o
o
o
o
o
o

Clinical Periodontology by Newman 8th & 9th Edition.

Diagnosis & Risk Prediction of Periodontal Diseases
Vol 3- Per Axelsson.
Periodontics Medicine, Surgery and Implants Rose
Outline of Periodontics - 4th edition JD Manson & Eley
Advances in Periodontics Wilson, Kornmn &
Newmann
Fundamentals of Periodontics- Wilson & Kornmn
Periodontology 2000; vol 34; 2004
British Dental Journal; vol 184: 1998.
Internet.
Presented by:
Sanjeeva kumar reddy C.M