Advanced diagnostic techniques

Raina J. P. Khanam
Dept. Of Periodontics
2nd year PG1

*
*Introduction
*Limitations of conventional periodontal diagnosis
*Advances in Clinical diagnosis
*Advances in Radiographic assessment
*Advances in Microbiologic analysis
*Advances in characterizing the Host response
*Conclusion
2

*
Definition of DIAGNOSIS (Dx):
*Diagnosis is defined as; correct determination,
discriminative estimation and logical appraisal of
conditions found during examination as evidenced by
distinctive marks, signs and characteristics of
diseases.
3

Conventional clinical diagnostic tools
*Periodontal diseases are prevalent human diseases
defined by the signs and symptoms of gingival
inflammation and periodontal tissue destruction.
4

*
1. Does not provide cause of the condition.
2. No information on patient’s susceptibility to the
disease.
3. Cannot identify sites with ongoing periodontal
destruction or sites in remission.
4. Cannot differentiate whether response to therapy is
positive or negative.
5

*
Advances in clinical diagnosis
Advances in radiographic assessment
Advances in microbiologic analysis
Advances in characterizing the host
response
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ADVANCES IN
CLINICAL
DIAGNOSIS
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1. Gingival bleeding
*Clinical evaluation of the degree of gingival inflammation includes
assessment of the redness and swelling of the gingiva along with
assessment of gingival bleeding.
*Although the earliest clinical signs of gingivitis consist of color
and texture changes, there may be underlying structural
alterations without corresponding clinical signs.
*Gingival bleeding is related to the persistent presence of plaque
on the teeth and is regarded as a sign of the associated
inflammatory response.
8

*Use of gingival bleeding as an indicator of inflammation has the
clinical advantage of being more objective, because color changes
require a subjective estimation.
*It has also been shown that gingival bleeding is a good indicator of
the presence of an inflammatory lesion in the connective tissue at
the base of the sulcus and that the severity of bleeding increases
with an increase in size of the inflammatory infiltrate.
*Clinicians tend to evaluate gingivitis by gingival bleeding alone, with
the use of a periodontal probe or a wooden interdental cleaner.
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*Absences of bleeding indicates periodontal stability.
*However, this fact may not be true in heavy smokers.
*Different studies have reported that tobacco smoking may
mask the inflammatory signs of gingivitis and periodontitis.
Lang et al. in a 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.
Lang et al. demonstrated that any force greater than 0.25 N may
evoke bleeding in healthy sites with an intact periodontium.
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2. Gingival temperature
*Commercially available system PerioTemp probe (Abiodent)
enables the calculation of temperature differential between
the probed pocket and subgingival temperature.
*It has two light indicating diodes:
 Red-emitting diode, which indicates higher temperature, risk for
future attachment loss.
 Green-emitting diode, which indicates low temperature, indicating
low risk.
Kung et al (1990) claim that thermal probes are sensitive diagnostic
devices for measuring early inflammatory changes in gingival tissue.
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*Possible explanation for increase in temperature with
increasing probing depth is an increase in cellular and
molecular activity caused by increased periodontal
inflammation.
Studies have demonstrated that the subgingival temperature at
diseased sites is increased compared with healthy sites and that a
natural antero-posterior temperature gradient exists within the
dental arches (posterior sites warmer than anterior sites).
In addition, mandibular sites were reported to be warmer than
maxillary sites.
Haffajee et al. also found that elevated subgingival site
temperature was particularly related to attachment loss in shallow
pockets and that Prevotella intermedia, Peptostreptococcus micros,
Porphyromonas gingivalis, Tannerella forsythia, and Actinobacillus
actinomycetemcomitans are also associated with elevated
temperatures.
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3. Periodontal probing
*The most widely used diagnostic tool for the clinical assessment of
connective tissue destruction in periodontitis is the periodontal probe.
Limitation of conventional probing
1) Lack of sensitivity and reproducibility.
2) Difference in measurement depends on: probing technique, probing
force, angle of insertion of probe, size of probe, precision of
calibration, presence of inflammation.
3) Readings of clinical pocket depth measured with probe does not
coinside with the histologic pocket depth.
4) All these variable contribute to the large standard deviations (0.5-1.3
mm) in clinical probing results.
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*Gabathuler and Hassell (1971) designed the 1st pressure
sensitive probe with a constant force application.
*Armitage (1977) designed another pressure sensitive probe to
determine how a constant probing pressure of 25pounds affected
the connective tissue attachment.
*Van der Velden (1978) designed pressure sensitive probe using
cylinder and piston connected to an air-pressure system. Their
device was capable of recording force alteration from 0.1N to
0.5N.
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*Tromp et al (1979) introduced a pressure sensitive probe in
which torque spring was attached to a loose probe head that
could rotate in a point bearing. They achieved a constant force
application of about 15gms.
*Polson (1980) introduced electronic pressure sensitive probe
known as Vine Valley probe. In this probe, handpiece and
control base was designed which allowed the examiner to
control the probing pressure.
*Frank hunter (1994) introduced true pressure–sensitive
probe, which have a disposable probing head and a hemispheric
probe tip with a diameter of 0.5mm. A rim surrounding the side
of the ball tip helps in detection of the cemento-enamel
junction, calculus or root surface irregularities.

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Limitations of 2nd generation probes
1. Constant pressure application
2. Errors in readings
3. Errors in the calculation of attachment loss
4. No automated recording system available
to store the data obtained

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Foster-Miller (Alabama probe)
*This probe was devised by Jeffcoat et al in 1986.
*Working at the university of Alabama, they were able to build a device
capable of providing controlled probing pressure and measuring the pocket
depth along with the detection of CEJ.
*The components of the probe included: a pneumatic cylinder, a linear
variable differential transducer (LVDT), a force transducer, an accelerator
and a probe tip.
*The detection of the CEJ is done by moving the ball tip of the probe on the
root surface at a controlled speed and preset pressure.
*When it reaches the CEJ there is an abrupt change in the acceleration
which is indicated by the graph, hence the CEJ is detected.

* Following these criteria of NIDCR, Florida
Probe System was developed by Gibbs et
al in 1988.
* The components of this probe includes, a
probe handpiece and sleeve, a displacement
transducer, foot switch, computer
interface.
* The hemispheric probe tip has a diameter of
0.4 mm and the sleeve has a diameter of
0.9mm.
* This probing system provides a constant
pressure of 15gms.
* It has 3 variants: the pocket probe, disk
probe and stent-based model.
* The second two probes use occlusal/incisal
surface for referencing.
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Toronto Automated Probe:
*McCulloch and Birek in 1991 at the University of Toronto described a
probe, like the Florida probe.
*This probe was incorporated with a tilt sensor device in its handle which
could identify changes in the angulation of the probe.
*The sulcus probing was done with a 0.5–mm nickel-titanium wire that is
extended under air pressure.
*Disadvantage was that: difficult in recording pocket depth around
second and third molars also patients have to position their heads in
same place.
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Interprobe:
*Also known as Perio Probe and uses fiber
optic technology.
*Goodson and Kondon (1988) used this
technology in their controlled-force Accutek
probe.
*Components of this system includes:
disposable probe tip, a control unit, memory
cards for recording data and foot switch.
*A fiber bundle transmits light to the
transducer and reflected light to a signal
processor.
*Probing depth is computed by comparison of
the reflected light signal with the reference
obtained from the zero position.

Peri Probe Comp:
*A computerized electronic probe with a controlled pressure of
0.4N in 2mm pockets and 0.2N in 13mm pockets.
*The components include a handpiece with disposable probe
sleeve unit and a small ball shaped 0.5mm diameter end.
*The handpiece contains a closed in spring, which controls the
probing pressure.
*The unit is connected to a computer which records the data
obtained by probing.
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Fourth generation periodontal probe:-
Periodontal probes utilizing 3D technology. These probes are
currently under development.
Fifth generation periodontal probe:-
Periodontal probes utilizing 3D technology and ultrasound.
A non invasive technique.
It was devised by Hinders et al (1999).
A very narrow beam of high-frequency (10-15MHz) ultrasonic waves
is directed into gingival sulcus.

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*While probing, the tip of the probe is kept vertically parallel to the
long axis of the tooth and placed gently on the gingival margin until
slight blanching of gingiva is visualized.
*Now the probe is activated with foot paddle to introduce a small
stream of water into the sulcus along with a thin beam of ultrasound
waves.
*The ultrasonic beam entering the tissues is either absorbed,
reflected or scattered.
*The reflected portion of the beam is received by the machine and
used for reconstruction of the ultrasonic image.
*The transducer installed in the handpiece records the echoes and the
computer analyzes the data obtained.

ADVANCES IN
RADIOGRAPHIC
ASSESSMENT
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*Dental Radiographs are traditional method to assess
destruction of alveolar bone.
*“Conventional radiographs are very specific but lack sensitivity”
*Primary criterion for bone loss is the distance from CEJ to the
alveolar crest and distance more than 2 mm is considered as the
bone loss.
*But variability affecting conventional radiographic technique
are:-
1) Variation in projection geometry.
2) Variation in contrast and density.
3) Masking by other anatomic structures.
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A. X–ray film taken without a
paralleling technique
showing a clear distortion of
the root length.
B. Image of the same tooth in A with a
proper image projection showing the
real alveolar bone height and
demonstrating severe bone loss in
the distal surface of the upper
first molar.
C. X–ray film taken without a paralleling
technique showing a clear distortion
of the root length relative to the
crown, The alveolar bone height is
obscured and fills the interproximal
space.
D. Image of the same tooth in C with a
proper image projection showing the
real alveolar bone height and the
open interproximal space.27

*The variations in projection geometry can be reduced by the use of
standardized long–cone paralleling radiographic techniques.
A. Position of the film holder relative to the teeth.
B. Position of the film holder relative to the paralleling device.
C. Position of the paralleling device to the x–ray long–cone tube.
D. Radiograph obtained with proper image projection.
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1. Digital Radiography
* The first direct digital imaging system, RadioVisioGraphy (RVG), was invented by
Dr. Frances Mouyens in 1989.
* The “Radio” part consists of a conventional x-ray generator connected to a highly
precise microprocessor timer for very short exposure times and an anatomically
adapted sensor with rounded edges and angles.
* The “Visio” part of the RVG unit stores the incoming signals during exposure and
then converts them point by point into one of 256 discrete grey levels.
* The “Graphy” part of the RVG unit consists of a digital mass storage unit that can
be connected to various video printout devices or direct photographs of the screen
may be made to provide an opportunity to access the radiographic information
further.
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Principles of digital radiography
* In the conventional film-based radiography, the film serves as both detector and
storage medium, whereas digital detectors are used only to generate the digital
image which is then stored on a digital medium.
* Digital imaging comprises 4 separate steps
1. Generation
2. Processing
3. Archiving
4. Presentation of the image
X-ray tube generates X-ray, which are then exposed to the sensor placed in the oral
cavity. The energy absorbed by the detector is then transformed into electrical
charges, which are then recorded, digitized and quantified into gray scale. The
software then processes the information provided by the sensor and converts the
raw data into clinically meaningful image. The final digital image is stored in
digitalized storage.

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There are 2 types of digital radiography technology:
1. Direct Digital Radiographic technology
2. Indirect Digital Radiographic technology

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Direct digital radiography technology
*This technology uses an X-ray generator and a solid state image
receptor, more commonly referred to as sensor.
*A sensor is made up of a silicon chip which is sensitive to light and has a
scintillator layer that converts X-rays to light.
There are 2 types of sensor:
1. CCD (charge couple device)
2. CMOS (complementary metal oxide semiconductor)

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CCD (charge couple device)
*A CCD chip is a metal oxide semiconductor (MOS) device.
*Its base is constructed of a material which is a good conductor under
certain conditions.
*The base layer is topped with a layer of metal oxide. Usually silicon is
used as the base material and silicon dioxide is used as the coating.
*The final top layer is also made of silicon-polysilicon.
*A CCD chip consists of an array of small cells or pixels. Inside these
cells are tiny photosensitive devices . These small devices are
designed such that they will behave like “buckets” that will collect
charge and hold it until it is drained out of the system.
*A pixel is the smallest component of an image. CCD pixel is approx.
40µ and the latest version is 20µ.

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CMOS (complementary metal oxide semiconductor)
*CMOS and CCD share common principles.
*In a CMOS chip, more of the electronic components controlling
the conversion of photon energy into the electronic signal are
incorporated into the chip itself.

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Indirect digital radiography technology
*This technology uses image plates having a detective layer of
photostimulable crystals that contain different halogenides such as
bromide, chlorine or iodine.
*The phosphor crystals are usually cast into plates and then into resin
material in an unstructured way.
*When X-rays are exposed on these photostimulable crystals, they
absorb their energy by bringing their electrons to higher energy
levels.
*These electrons may stay at a higher energy level for several hours
depending on the specific physical properties of the phosphor crystals
used.
*The next step involves scanning of the image plate with laser beam.
*This converts the energy stored in the photostimulable crystals into
light.
*An array of photomultipliers collects the light, which is converted into
electrical charges by an analogue to digital convertor.
*The digitally converted raw information in the form of electrical
charges is now converted into a meaningful image by the software.

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ADVANTAGES OF DIGITAL RADIOGRAPHY
1. Reduced exposure as compared to conventional films.
2. A superior gray scale resolution of 256 shades of gray in comparisons
to 16-25 shades of gray on a conventional film.
3. Colorization and enlargement of the images can be done.
4. Decreased processing time.
5. Storage of radiographs into the small hard drive of the computer is a
great space saver.
6. Digital images can be easily transmitted to other dental offices.
7. It is also environment friendly because there is no disposal hazards of
processing chemicals, silver salts in film emulsion and lead foil sheets.
8. Beneficial during implant placement.

2. Subtraction Radiography
*Subtraction radiography was introduced by Ziedses des Plantes (1934).
*This technique requires a paralleling technique to obtain a standardize
geometry and accurate super imposable radiographs.
*This technique facilitates both quantitative and qualitative visualization
of even minor density changes in the bone.
*Bone gain appears as light areas and bone loss appears as dark areas.
ADVANTAGES
1. a high degree of correlation between changes in alveolar bone and CAL
after periodontal therapy.
2. increased detectability of small osseous lesions.
DISADVANTAGE
1. The need to be almost at identical projection alignment during the
exposure of the sequential radiographs, which makes this method
impractical in a clinical setting.
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Modification to subtraction methods,
called digital 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.
Webber et al (1982) and Grondahl
et al (1983) introduced digital
subtraction into dental radiography.
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3. Computer-Assisted Densitometric
Image analysis System
*In the computer-assisted densitometric
image analysis system (CADIA), a Video
camera measures the light transmitted
through a radiograph and the signals from
the camera are converted into gray scale
images.
*CADIA appears to offer an objective
method for alveolar bone density changes
quantitatively over time.
*Also, compared with digital subtraction
analysis, CADIA has shown a higher
sensitivity and a high degree of
reproducibility and accuracy.
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4. Absorptiometry
* It is a non invasive method which can detect even small bone density changes.
* Introduced by Henrikson (1976) it is the most precise method to detect periodontal
bone mass changes.
* It is based on the principle of photon absorptiometry.
* The tissue composition is assessed by the differential absorption of gamma rays (or X-
rays) by different tissues such as bone, fat and other soft tissue.
* The source of gamma rays is generated from radioisotopes such as gadolinium, iodine,
americium.
* 3 kinds of technique described in photon absorptiometry:-
1. Single-photon absorptiometry (SPA)
2. Dual-photon absorptiometry (DPA)
3. Dual energy X-ray absorptiometry (DEXA)

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5. Computed Tomography
*The word ‘tomography’ comes from the greek word
‘tomos’ means slice and ‘graphien’ means ‘to write’.
*In 1972, an English engineer GN Hounsfield built
the first commercial, CT scanner.
*It is the technique of reconstructing a cross-
sectional image of the body from a virtual pile of X-
ray photographs.
*The patient remains stationary on the examination
table while the X-ray tube rotates in a circular orbit
around the patient.
*It is divided into 2 categories:-
1. Fan beam
2. Cone beam
*In fan beam scanners, data is acquired using a narrow
fan-shaped X-ray beam transmitted through the
patient.

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6. Cone-Beam Computed Tomography
*Also known as Cone Beam Volumetric Tomography (CBVT) refers to a
tomographic imaging beam that is concentrated in a narrow field of the
body.
*Multi-dimensional images of the hard tissue of the jaws can be obtained
using this technology.
*CBCT provides an image of hard tissue that has no distortion and is
anatomically correct.
*This is combined with a 3D X-Ray beam.
*It involves a single 360 scan in which the X-ray source and a reciprocating
area detector moves around the pt. head which is stabilized with a head
holder.
ADVANTAGE
In the diagnosis of implant case,
providing clear images of highly
contrasted structures and for
evaluating bone.
Reduction of X-ray dosage.

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7. Magnetic Resonance imaging
*MRI is a widely used diagnostic tool in the medical field.
*This technique was first introduced by Lauterbur in 1972.
*The phenomenon of magnetic resonance results from the dynamics of
molecular spins in combined static and oscillating magnetic fields.
*Now here, magnetic needles in our body are the nuclei of the hydrogen
molecules, components of water molecules present abundantly in our body.
*In MRI scan the weak push to these molecules is given by placing magnets
in a perpendicular direction and moving them towards and away from these
nuclei.
*The radio waves emitted are recorded and the data is interpreted to make
3 dimensional image of the region under investigation.
ADVANTAGE
1. Ability to demonstrate the soft tissue
2. Also can locate inferior alveolar canal
3. Better visualization of peri-implant tissue

ADVANCES IN
MICROBIOLOGIC
ANALYSIS
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Purpose of microbiologic analysis
1. Support diagnosis of various Periodontal disease
2. Can tell about initiation & progression
3. To determine which periodontal sites are at high
risk for active destruction
4. Can also be used to monitor Periodontal therapy
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Sample Collection
A and B, Tooth is dried, and supragingival
plaque is removed from the sampling site.
C, Three sterile endodontic paper points
are placed sub-gingivally to the base of
the pocket.
D, The paper points are removed and
placed in a vial of anaerobic media for
immediate transport to the laboratory for
analysis.
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1. Bacterial Culturing
Historically, culture methods have been widely used in studies aimed at
characterizing the composition of the subgingival microflora and are still
considered the reference method (“gold standard”) when determining the
performance of new microbial diagnostic methods.
Advantage
1. The clinician can obtain relative and absolute counts of the cultured
species.
2. The only in vitro method able to assess for antibiotic susceptibility of
the microbes.
Disadvantage
1. Culturing requires sophisticated equipment and experienced personnel.
2. Time–consuming
3. Expensive
4. Can only grow live bacteria (lactobacillus)
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2. Direct Microscopy
*Dark–field microscopy has been suggested as an alternative to culture
methods on the basis of its ability to assess directly and rapidly the
morphology and motility of bacteria in a plaque sample.
*However, most of the main putative periodontal pathogens, including Aa,
Pg, Tf, Eikenella corrodens (Ec), and Eubacterium species, are non-motile,
and therefore this technique is unable to identify these species.
*Therefore, dark–field microscopy seems an unlikely candidate as a
diagnostic test of destructive periodontal diseases.
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*Fluorescence microscopy, a fluorochrome is excited by ultra
violet light, resulting in visible fluorescence. As a result, a
bright image, in a dark background is seen.
*It is used extensively to study the intracellular distribution,
dynamics and molecular mechanisms of a large variety of
macromolecules and metabolites.

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3. Chromatography
*Various periodontal pathogens produce metabolites, including volatile
compounds like volatile sulphur compounds.
*Bacteria like Treponema denticola, Porphyromonas gingivalis, Prevotella
intermedia and Tannerella forsythia are capable of producing sulphates.
*They degrade serum proteins, cysteine and methionine resulting in the
production of measurable amounts of Sulphur, Hydrogen Sulphide, Methyl
Mercaptan and dimethyl sulphide.
*These resultant volatile sulphur compounds can be detected biochemically by
chromatography.
*Chromatography involves 2 phase:
1. Mobile phase
2. Stationary phase
*The mobile phase (gas or liquid) contains and carries the sample to be
analyzed through or across the stationary phase. The stationary phase
maintains conditions necessary for separating various substances (analytes)
within the sample being studied.

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Diamond Probe/Perio 2000 System
*A sulfide sensor, Perio 2000 can measure the levels of sulfide
compounds and report them as scores ranging from 0 to 5.
*This test is a non-specific test which cannot identify the bacteria
responsible for the production of these sulfide compounds, but
can still predict sites infected with periodontal pathogenic
bacteria.

ADVANCES IN
IMMUNODIAGNOSTIC
ANALYSIS
52

Immunodiagnostic Methods
*Immunologic assays employs antibodies that recognize
specific bacterial antigens to detect target microorganisms.
Variety of procedures used:-
1. Direct (DFA) and indirect (IFA) immunofluorescent assays
2. Radio-immunoassay (RIA)
3. Flow cytometry
4. Enzyme-linked immunosorbent assay (ELISA)
5. Latex agglutination
53

1. Direct IFA employs both monoclonal and polyclonal
antibodies conjugated to a fluorescein marker [such as
fluorescein isothiocyanate (FITC) or tetramethylrhodamine
isothiocyanate (TRITC)] 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.
IFA has been used mainly to detect Aa and Pg.
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2. Radio-immunoassay (RIA)
*With this technique, any biological substance for which a specific antibody
exists can be measured, even in minute concentrations.
*The procedure of RIA consists of adding suitable quantities of standard,
unknown, radio-labelled antigen and antibodies to a buffer solution and
allowing the reaction to reach equilibrium. For radiolabelling, Iodine labels are
commonly used.
*Unlabeled antigen are added to the samples of the mixture.
*These compete for the binding sites of the antibodies.
*At the end of the incubation period, the free and bound fractions are
separated using a suitable technique.

3. Cytofluorography or flow cytometry
The basic components flow cytometry are:
1. Fluidics
2. Optics
3. Electronics
The fluidic system transports particles in a stream to the laser
beam.
The optical system illuminates the particles for detection of the
resultant light signals by optical filters.
The electronic system converts the detected light signals into
electronic signals that can be processed by the computer.
For the 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.
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4. Enzyme–linked immunosorbent assay
(ELISA) is similar in principle to other
radio-immunoassays, but instead of the
radioisotope, an enzymatically derived color
reaction is substituted as the label.
*The intensity of the color depends on the
concentration of the antigen.
*ELISA has been used primarily to detect
serum antibodies to periodontal pathogens.
A membrane immunoassay has been adapted for chair-
side clinical diagnostic use (Evalusite), developed by
Eastman Kodak.
It involves linkage between the antigen and a membrane-
bound antibody to form an immuno-complex that is later
revealed through a colorimetric reaction.
Evalusite has been designed to detect Aa, Pg, and Pi
within 8minutes. 57

5. Latex agglutination is a simple immunologic 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; agglutination or clumping is then visible,
usually in 2 to 5 minutes.
Because of their simplicity and rapidity, these assays have great potential
for chair side detection of periodontal pathogens.
However, these assays have only been tested for research purposes and are
not clinically available.
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ADVANCES IN
MOLECULAR BIOLOGY
ANALYSIS
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*The principles of molecular biology technique reside in the analysis of
DNA, RNA and the structure and function of proteins.
*Molecular diagnostic tests can be broadly classified into:-
1. Hybridization
2. Amplification
3. Sequencing
4. Enzymatic digestion of nucleic acids
1. Nucleic acid probes
A probe is a known, single stranded nucleic acid molecule (DNA or RNA)
from a specific pathogen synthesized and labeled with a enzyme of a
radio isotope.
Hybridization: Pairing of complimentary strands of DNA to produce a
double stranded DNA.
DMDx and Omnigene are commercially available genomic probes for the
detection of Aa, Pg, Pi and Td.

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2. Checkerboard DNA-DNA hybridization
technology
*Socransky et al. in 1994 developed this
technique for the detection and levels of
40 bacterial species often found in the
oral cavity.
*The method requires sophisticated
laboratory equipment and expertise and
is highly specific, and thus this assay has
not been generalized for diagnostic
purposes.
*It is particularly applicable, however, for
epidemiologic research and ecologic
studies because it does not require viable
bacteria and allows for the assessment
of large number of plaque samples and
multiple species.

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3. Polymerase chain reaction (PCR)
*Polymerase chain reaction (PCR) has emerged as the most powerful tool for
the amplification of genes and their RNA transcripts.
*This technique, developed in 1985, is the single technique used almost
universally to study DNA and RNA obtained from a variety of tissue
sources.
*PCR typically begins with the isolation of DNA from a fresh tissue
specimen. By heating the complementary double strands, DNA splits into
single–stranded. The amplification is followed using a DNA polymerase that
requires a primer. Taq polymerase is the commonly used primer at 72C.
For obtaining amplified fragments of constant length and in large
quantities, a second primer, must be used to anneal (bind). This
amplification can be performed several times, known as cycles. This
sequenced DNA is then detected and visualized through electrophoresis in
agarose gel and ethidium-bromure, obtaining a qualitative signal.
*MicroDent Test is the chair side
diagnostic kit.

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*Various derivatives of PCR procedure are available today such as:-
1. Multiplex PCR
2. Nested PCR
3. Quantitative PCR
4. Reverse transcription PCR
5. Arbitrary primed PCR
6. Real time PCR
Multiplex PCR:- More than one primer pair is included in the PCR mixture.
For eg; in the detection of a particular bacteria, one primer pair
can be directed at sequences present in relevant bacteria.
Second primer pair can be directed at a sequence specific for a
particular gene.
Nested PCR:- this involves the sequential use of two primer sets. The
initial primer set is used to amplify a target sequence. The
amplicon obtained is then used as the target sequence for a
second amplification using primers internal to those of the first
amplicon.

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Quantitative PCR:- As the name indicates, this PCR procedure has
the ability to quantitate the actual number of targets present
originally in the clinical specimen. The procedure monitors the
accumulation of a DNA product from PCR reaction.
Reverse transcription PCR:- It is very useful modification of the
basic PCR procedure because many clinically important viruses have
genomes composed of RNA rather than DNA (eg. HIV, hepatitis B
virus). The unique step to this procedure is the use of the enzyme
reverse transcriptase that directs the synthesis of DNA from the
viral RNA template. Once the DNA has been produced, routine PCR
technology is applied to obtain amplification. Comercial chairside
diagnostic kit is MyPerioPath.

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Arbitrary primed PCR:- In this PCR procedure, short
primers are used for amplification which is not specifically
complementary to a particular sequence of a target DNA.
After completion of the reaction, multiple annealing sites
result in the amplification of multiple fragments of
different sizes.
Real time PCR:- It is a recent modification to PCR. It allows
precise quantification of specific nucleic acids in a complex
mixture even if the starting amount of material is at a
very low concentration. This is accomplished by rapid
thermocycling and monitoring the amplification of a target
sequence in real time using fluorescent technology. It is
capable of detecting the presence of a target within 30 to
120minutes.

4. Enzymatic Methods
*Tannerella forsythia (Tf), Porphyromonas gingivalis
(Pg), the small spirochete Treponema denticola
(Td), and Capnocytophaga species share a common
enzymatic profile: all have a trypsin like enzyme.
*The activity of this enzyme can be measured with
the hydrolysis of the colorless substrate N–
benzoyl–d L–arginine–2–naphthylamide (BANA).
*When the hydrolysis takes place, it releases the
chromophore β -naphthylamide, which turns orange
red when a drop of fast garnet is added to the
solution.
*Diagnostic kits have been developed Perioscan.
66

67
*Loesche 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.
*Loesche also showed that shallow pockets exhibited only 10%
positive BANA reactions, whereas deep pockets (7 mm)
exhibited 80% to 90% positive BANA reactions.
A subgingival plaque sample obtained from the patient is placed on
the BANA containing strip which is then folded to contact a second
strip containing the “Fast black” dye reagent. It is then placed in an
oven for 15min at 55C. Identification of any blue-black color on the
strip indicates the presences of trypsin like protease in the plaque
sample.

ADVANCES IN
CHARACTERIZING
THE HOST
RESPONSE
68

*Assessment of host response refers to the study of
mediators by immunologic or biochemical methods, that
are recognized as a part of individual’s response to the
periodontal infection.
69
Inflammatory mediators and products
Host derived enzymes
Tissue breakdown products

1. Source of samples
* Sample sources include saliva, gingival crevicular fluid (GCF), gingival crevicular
cells, blood serum, blood cells, and urine.
* For the collection of GCF, a number of approaches have been used, ranging from
paper strips, to micro–capillary tubes and micropipettes, to micro syringes and
plastic strips.
* Saliva can be collected from the parotid, submandibular and sublingual glands.
70
The most common method is the use of
perio-paper strips. These strips are
placed in the gingival sulcus for a
standard time until the filter paper is
saturated. The fluid volume collected on
the strips can be then quantified in a
number of ways. At present, the most
common way is using the Periotron device.

2. Inflammatory mediators and Products
Cytokines present in GCF and investigated as potential diagnostic markers
includes:
 Tumor Necrosis Factor alpha (TNF )
 Interleukin-1 (IL-1)
 Interleukin-1 (IL-1)
 Interleukin-6 (IL-6)
 Interleukin-8 (IL-8)
Prostaglandin E2 (PGE2) is a product of the cyclooxygenase pathway of the
metabolism of arachidonic acid. It is a potent mediator of inflammation
and induces bone resorption.
71

3. Host-Derived Enzymes
The breakdown of collagen occurs during inflammation by two
different pathways:-
1. Intracellular
2. Extracellular
Intracellular destruction enzymes are
 Aspartate amino-transferase
 Alkaline phosphatase
 β-glucuronidase
 Elastase.
Extracellular is associated with the activity of matrix
metalloproteinases.
72

Aspartate aminotransferase (AST) is an enzyme released from
dead cells from a variety of tissues throughout the body,
including the heart (after myocardial infarction) and the liver
(during hepatitis).
*A marked elevation in AST levels in GCF samples from sites
with severe gingival inflammation have been seen.
*A commercial chair side test kit for AST has been developed
(PerioGard). The test involves collection of GCF with a filter
paper strip, which is then placed in tromethamine
hydrochloride buffer. A substrate reaction mixture containing
L-aspartic and α–ketoglutaric acids are added and allowed to
react for 10 minutes. The addition of a dye, such as fast red,
results in a color product.
*Pocket watch chair side diagnostic kit has been designed for
the detection of levels of AST in GCF. The biochemical
principle behind this test is that AST catalyzes the transfer
of an amino group of cysteine sulfuric acid by -keto-glutaric
acid to yield -sulfinyl pyruvate in the presence of pyridoxal
phosphate.
73

*Alkaline phosphatase (ALP) is an enzyme found in many cells of
the periodontium, including osteoblasts, fibroblasts, and
neutrophils.
*Cross–sectional studies show that concentrations of this
enzyme in GCF from diseased sites are significantly higher than
from healthy sites.
74
β–Glucuronidase (βG) is a lysosomal enzyme found in the primary
(azurophilic) granules of neutrophils. A diagnostic kit based on -
glucuronidase is being commercially developed by Abbott
Laboratories, North Chicago, USA. It is a color detection system
which will probably uses a histochemical substrate.

75
*Perio Check:- This system detects the presences of neutral
proteases such as collagenase in GCF. GCF is obtained in a paper
strip which is placed on a gel containing insoluble dye-labelled
collagen fibrils and incubated at 43C. If neutral protease is
present in GCF, they diffuse in the gel and the insoluble collagen
dye complex is digested to release soluble dye labelled
fragments, which diffuse back into the strip, turning it blue.
The intensity and the area of the blue color are then stored on
a scale of 0 to 2 by comparing it with three standards on a color
card which is provided with the test kit. The intensity of the
color is proportional to the amount of enzyme present in the
sample.

76
Elastase is a serine protease also stored in the primary granules
of neutrophils. Prognos-Stik kit is designed for the detection
of serine protease, elastase in GCF samples. A GCF sample is
collected on special paper strips which have been impregnated
with the appropriate peptidyl derivative of 7-amino-
trifluromethyl coumarin (AFC). If elastase is present, the
sample reacts with the substrate in 4-8minutes releasing the
fluorescent. This produces green fluorescence in the strip
which can be seen under UV light using a UV light box. The
intensity of fluorescence is directly proportional to the amount
of elastase present in the GCF.

*Matrix metalloproteinases (MMPs) are members of a large
subfamily of zinc–dependent and calcium–dependent proteolytic
enzymes (proteinases) responsible for remodeling and
degradation of extracellular matrix components.
*MMPs are release by different cells, such as fibroblasts and
macrophages, and the presence of tissue inhibitors of MMPs
(TIMPs) that are widely distributed in tissues and fluids.
*MMP-8 plays an important role in periodontal tissue destruction.
*A chairside test kit has been developed for detection of MMP-8
is Dip Stick Test.
77

4. Tissue Breakdown Products
The connective tissues of the periodontium are composed of
1. Fibrous proteins such as:-
 Collagen
 Elastin
2. non-fibrous glycoproteins such as:-
 Laminin
 Fibronectin
 Proteoglycans
3. Minerals
4. Lipids
5. Water
6. Tissue–bound growth factors.
The extracellular matrix of the periodontium include
a. Proteoglycan (versican, decorin, biglycan, syndecan)
b. noncollagen proteins (elastin, fibronectin, laminin, osteocalcin, osteopontin,
bone sialoprotein, osteonectin, tenascin).
78

Although there are many potential markers for
periodontal disease activity and progression,
numerous features still hamper the ability to use
them as diagnostic tests of proven utility. After all
these years of intensive research, we still lack a
proven diagnostic test that has demonstrated high
predictive value for disease progression.
79

Advanced diagnostic techniques

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