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RECENT ADVANCES IN PERIODONTAL DIAGNOSIS
PRESENTED BY:SONIA SACHDEVA
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Diagnosis is the art of distinguishing one disease from another and the determination of the nature, location and causes of a disease.
Periodontal diagnosis is defined as “recognizing a departure from normal in the periodontium and distinguishing one disease from another. It must be based on compilation of information from the medical and dental history, from symptoms, from signs of disease and from clinical and radiographic examination of the patient.”
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GENERAL PURPOSE OF PERIODONTAL DIAGNOSTIC PROCEDURES
Screening
Diagnosis of specific periodontal diseases.
Identification of sites or subjects at an increased risk of experiencing the progression of periodontal destruction.
Treatment planning
Monitoring of therapy.4
CONVENTIONAL DIAGNOSTIC AIDS
Clinical diagnosis- Clinical signs of inflammation
Bleeding on probing Increased GCF Pocket depth Attachment level
measurements Radiographic diagnosis-IOPA Bitewing Panoramic
radiography Microbiological diagnosis – Culturing
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LIMITATIONS OF CONVENTIONAL TECHNIQUES Conventional diagnostic techniques do not reliably differentiate sites with ongoing periodontal destruction
and inactive sites provide information on the patient’s susceptibility to disease differentiate disease is progressing or in remission
Periodontal disease is site specific and has a multifactorial origin where periodontal pathogens, host response, genetic, systemic, and behavioral risk factors interplay to develop the disease process Multifactorial origin of periodontal disease
Consideration should be given to include microbiologic, immunologic, systemic, genetic, and behavioral factors, in addition to the traditional clinical and radiographic parameters6
Clinical Diagnosis
Gingival Temperat
ure
Periodontal Probing
Tooth mobIlity
Oral malodor
Occlusal analysis
Radiographic assessment
Digital radiograp
hySubstracti
on radiograp
hy
CADIA
Computed
tomography
Microbiologic analysis
Bacterial culturing
Direct microscop
y
Immunodiagnostic
Enzymatic Diagnosti
c assays based on molecular
biology
Characterizing Host
response
Inflammatory
mediators and
Products
Host derived
enzymes
Tissue breakdow
n Products
Others
Genetic diagnosis
Oral rinse test
Advances in
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ADVANCES IN CLINICAL DIAGNOSIS
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PERIODONTAL PROBING
Increased probing depth and loss of clinical attachment are pathognomonic for periodontitis.
Pocket probing is a crucial and mandatory procedure in diagnosing periodontitis and evaluating periodontal therapy.
Reduction of pocket depth and gain of clinical attachment are the major clinical outcome measurements used to determine success of treatment
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Limitations of conventional Probes: Lack of sensitivity and reproducibility of the measurements. Readings of clinical pocket depth obtained with the periodontal
probe do not normally coincide with the histologic pocket depth, since the probe normally penetrates the coronal level of the junctional epithelium, and the precise location of the probe tip depends on the degree of inflammation of the underlying connective tissues.
The disparity between measurements also 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.
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NIDCR CRITERIALimitation Conventional NIDCR Criteria
Precision 1mm 0.1mm
Range 12mm 10mm
Probing Force Non-standardized Constant
Applicability Noninvasive Noninvasive
Reach Easy to access Easy to access
Angulation Subjective Guidance System
Readout Voice dictation and recording
Direct electronic reading
Security Easily sterilized Complete sterilization
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GENERATIONS OF PERIODONTAL PROBES
Type CharacteristicsFirst Generation: conventional probe ;manual probes.
Usual clinical instrument : a thin tapering tine marked to be read in mm.
Second generation: constant force probes. Pressure sensitive probe.(given by Vander Velden 1978)
As above, but with a spring or electronic cut-out when the appropriate fore is reached. Force 30g probe tip remains in the CEJ and force of 50gm are necessary to diagnose osseous defects .e.g. vine valley; vivacare TPS
Third generation : automated probes When probe is in place with specified force, a device is activated that reads the measurement accurately. Automated and computerized probe e.g.Florida ;Foster miller ; automated
Fourth generation : Three-dimensional probes
These are aimed at recording sequential probe positions along a gingival sulcus
FiFth generation : non-invasive three dimensional probes
These will add ultrasound or another device to a fourth generation probe.Identifying attachment level with and penetrating it .
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FIRST GENERATION PROBES
are manual probes like William’s Periodontal probe UNC 15 probe CPITN Probe Michigan Probe etc.
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Williams probe has markings at 1, 2, 3, 5, 7, 8, 9, and 10 mm.
Marquis probe or Hu-Friedy color coded probe is color coded by alternately colored or black and silver bands that mark 3, 6, 9, and 12 mm.
Michigan O probe with Williams markings has circumferential lines at 1 mm, 2 mm, 3 mm, 5 mm, 7 mm, 8 mm, 9 mm, and 10 mm.
PCP12 probe with Marquis markings has alternating shades every 3 mm.
CPITN probe has markings at 3 5, 5.5, 8.5, and 11.5 mm.
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SECOND GENERATION PROBES (PRESSURE SENSITIVE)
Objective - to reduce measurement variation by standardizing probing force.
Probes available : Gabathuler probe True Pressure-sensitive probe (Vivacare) Prockprobe PDT Sensor Probe Vine Valley probe
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TRUE PRESSURE SENSITIVE (TPS) PROBE Prototype for second-generation probes.
Introduced by Hunter in 1994 a disposable probing head and a
hemispheric probe tip with a diameter of 0.5 mm.
controlled probing pressure of 20 gm These probes have a visual guide and a
sliding scale where two indicator lines meet at a specified pressure.
is increased unti
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In 1977, Armitage designed a pressure-sensitive probe holder to standardize the insertion pressure and determine how accurate probing pressure of 25 pounds affected the connective-tissue attachment.
In 1978, Vander Velden devised a pressure-sensitive probe with a cylinder and piston connected to an air-pressure system. Subsequently, it was modified with a displacement transducer for electronic pocket-depth reading. 17
VINE VALLEY PROBE
Introduced by Polson et al 1980
It is an electronic pressure sensitive probe that is not sensitive to lateral forces and not subjected to error due to gravity.
It allows control of insertion pressure & permits the use of different types of probe tips.
The pressure force varies with a range of sensitivity of 5-100gms
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THIRD GENERATION PROBES (AUTOMATED/COMPUTER LINKED
ELECTRONIC CONSTANT PRESSURE)
The first controlled probing force probe that was capable of automated detection of the CEJ for determination of attachment levels and allowed for computerized data recording was presented by Jeffcoat et al (1986).
Probes available:
Florida probe Toronto probe Foster-Miller ( Alabama) probe Interprobe
Insertion forces have in vitro resolution of 0.1-0.5mm.
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FLORIDA PROBE
designed by Gibbs et al (1988). Advantage : Constant probing force Precise electronic measurements Computerised data capturing Complete sterilization
Limitations lack tactile sensitivity fixed-force setting throughout the mouth underestimation of deep probing depths
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Two versions of the Florida Probe® handpiece are available for the determination of relative attachment levels: the stent probe and the disk probe.
Stent probe uses an acrylic stent as a reference. Disk probe has a small metal disk attached to the
sleeve and uses the occlusal surface or incisal edge of a tooth as a reference.
measurements made electronically and transferred automatically to the computer when the foot switch is pressed.
Constant probing force is provided by coil springs inside the probe hand piece and digital readout.
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FOSTER MILLER PROBE
Devised by Jeffcoat et al in 1986, this probe has controlled probing pressure
can automatically detect the position of the CEJ
record the clinical attachment level is the prototype of third-generation probes. The components of the probe are: a
pneumatic cylinder, a linear variable differential transducer (LVDT), a force transducer, an accelerator, and a probe tip.
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TORONTO PROBE
McCulloch and Birek (1987) designed this probe in the university of Toronto (Canada).
Uses the occlusal-incisal surface as a reference point to measure the clinical attachment level.
The sulcus is probed with a 0.5-mm nickel-titanium wire that is extended under air pressure.
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INTERPROBE
Calibrated for a constant 0.3-N (1.26 N/mm2) probing force and uses a 0.55-mm-diameter plastic filament.
It is based on fiber optic technology. The probe tip is attached to an optical
encoder transducer element. 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.
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FOURTH GENERATION PROBES (ULTRASONIC ) Projects a very narrow beam of high-frequency
(10-15 MHz) ultrasonic waves into the gingival sulcus.
Detects echoes of returning waves, which are reflected back from tissues.
The focused ultrasonic beam is transmitted into the sulcus in the same orientation as a manual probe.
Ultrasound probe tip is gently placed on the gingival margin until slight blanching occurs, then swept along the entire gingival area.
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Advantages- detects much smaller increments of
anatomic change earlier detection of tissue breakdown additional histological information, such
as tissue thickness and inflammation permits earlier diagnosis and
intervention Handpiece design is ergonomically
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FIFTH-GENERATION PROBES Despite all the advances in earlier generation
probes, they remain invasive and, at times, their use can be painful to patients.
With these earlier generation probes, the probe tip usually crosses the junctional epithelium.
Fifth-generation probes are being devised to eliminate these disadvantages.
Probes are being designed to be 3D and noninvasive: an ultrasound or other device is added to a fourth-generation probe
Fifth-generation probes aim to identify the attachment level without penetrating it.
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UltraSonographic (US) probe uses ultrasound waves to detect, image, and map the upper boundary of the periodontal ligament and its variation over time as an indicator of the presence of periodontal disease. It was devised by Hinders and Companion at the NASA Langley Research Center.
This small intraoral probe has an ultrasound beam projection area close enough in size to the width of the periodontal ligament space to give the optimal coupling and small enough to inspect the area between the teeth, while still delivering sufficient signal strength and depth of penetration to image the periodontal ligament space.
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The Ultrasonic Periodontal Probe
To probe structures ultrasonically, a narrow beam of ultrasonic energy is projected down between the tooth and bone from a transducer, which is scanned manually along the gingival margin.
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The ultrasound transducer is mounted in probe-tip shell, which incorporates a slight flow of water to ensure good coupling of the ultrasonic energy to tissues.
The couplet water can come either from a suspended intravenous-type sterile bag or plumbed from the dental-unit water source.
The focused ultrasonic beam is transmitted into the pocket in the same orientation as the insertion of a manual probe
Then, the probe is moved along the gingival margin, so the two-dimensional graphical output corresponds to the results a clinician gets from “walking the sulcus” with a manual probe.
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However, ultrasound gives more information because secondary echoes are recorded from tissue features at various depths. It appears likely that the technique also will be able to provide information on the condition of the gingival tissue and the quality and extent of the epithelial attachment to the tooth surface.
This may supply valuable data to aid the clinician in the diagnosis and treatment charting of these diseases
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CALCULUS DETECTION
Calculus detection probes detect subgingival calculus by means of audio readings and are reported to increase chances of subgingival calculus detection.
DetecTar (DENTPLY)- has a lightweight, well-balanced handpiece, which can be autoclaved, and it produces an audible beep to signify calculus detection (beep function can be disengaged). This probe may augment standard methods of calculus detection.
Expensive and the handpiece is bulkier than a standard periodontal probe.
As with many automated probes, there is potential for false positives and false negatives.
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AUTOMATED CALCULUS-DETECTION TECHNOLOGIES
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GINGIVAL TEMPERATURE
PerioTemp® probe detects pocket temperature differences of 0.1° C from a reference subgingival temperature.
Individual temperature differences are compared with those expected for each tooth, and 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.
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PERIODONTAL DISEASE EVALUATION SYSTEM Diamond Probe/Perio 2000 System reportedly
detects periodontal disease during routine dental examinations by measuring relative sulfide concentrations as an indicator of gram-negative bacterial activity.
Consists of a single-use disposable probe tip with microsensors connected to a main control unit.
Might detect periodontal disease at an early stage and might find an active site that requires treatment.
However, the probing pressure is not controlled. Also, periodontal disease can be caused by bacteria that do not produce volatile sulphur compounds, creating the potential for some disease activity to be missed.
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DIAGNOSING TOOTH MOBILITY
Periotest System (Schulte et al. 1992)
The use of the Periotest, a non-invasive, electronic device that provides an objective measurement of the reaction of the periodontium to a defined impact load.
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PERIOTEST SCALE
-8 to +9: clinically firm teeth (Normal tooth mobility)
10 to 19: first distinguishable sign of movement (Mobility 1)
20 to 29: crown deviates within 1 mm of its normal position (Mobility 2)
30 to 50: mobility is readily observed (Mobility 3)
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LASER VIBROMETER METHOD
Dynamic loads are applied and measured on teeth, with a small hammer and a load cell.
The consequent displacement of tooth is then measured with a laser Doppler vibrometer
Ratio between the maximum of the tooth displacement and the input force peak are considered as the mobility degree index.
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RESONANCE FREQUENCY ANALYSIS
This method evaluates the stiffness of the bone-implant interface by means of a signal transducer connected to a frequency response analyzer (Osstell; Integration Diagnostics, Göteborg, Sweden).
Osstell displays the peak of a frequency-amplitude plot.
Resonance frequency of the transducer-implant unit is calculated.
Implant stability quotient (ISQ) is displayed as a number between 1 and 100.
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DENTAL MOBILITY CHECKER (DMC)
Originally developed by Aoki and Hirakawa. Measures tooth mobility with an impact
hammer method using transient impact force. Aoki and Hirakawa successfully detected the
level of tooth mobility by converting the integration (rigidity) of tooth and alveolar bone into acoustic signals.
A microphone is used as receiver and response signal transferred from the microphone is processed by fast fourier transform (FFT) for conversion for analysis in the time axis.
Hence, the duration of the first wave generated by impact is detected. 42
DMC uses a small impact hammer as an excitation device. It is easily used even in molar regions.
DMC may provide quite stable measurement for osseointegrated implants.
But application of a small force to an implant immediately after placement may jeopardize the process of osseointegration.
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ADVANCES IN DIAGNOSIS OF ORAL MALODOR
Portable Sulfide Meter (Halimeter®) uses an electrochemical, voltametric sensor
which generates a signal when it is exposed to sulfide and mercaptan gases and measures the concentration of hydrogen sulfide gas in ppb.
portable and does not require skilled personnel for operation.
Disadvantages -necessity of periodic re-calibration and the measurements cannot be made in the presence of ethanol or essential oils. In addition, this limitation does not allow the assessment of mouthwash efficacy until after these components have been thoroughly rinsed out or dissipated.
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ELECTRONIC NOSE
a hand held device, being developed to rapidly classify the chemicals in unidentified vapor.
Its application by scientists and personnel in the medical and dental field as well as it is hoped that this technology will be inexpensive, miniaturizable and adaptable to practically any odor detecting task.
This device is based on sensor technology that can smell and produce unique fingerprints for distinct odors.
Preliminary data indicates that this device has a potential to be used as a diagnostic tool to detect odors.
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B/B CHECKER®
As a single unit, this device is capable of detecting several kinds of gases mixed with VSCs in addition to other odorous gasses.
Naofumi Tamaki (2011)- evaluated the effectiveness of the B/B Checker® for detecting the malodor level of oral, exhaled, and nasal air.
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Jayaram S (2010) developed a fluorimetric assay, using fluorescein mercuric(II) acetate (FMA), for the quantification of VSC in mouth air.
The assay is based on the quenching of fluorescence of FMA on reaction with VSC. The detection limit of the sensor is 0.06 μg L−1 for VSC. The analyte concentration, the pH of solution and the time for reaction have been optimized to achieve rapid and sensitive response for VSC levels in the range of 0–0.75 μg L−1. 48
The response of the sensor has been validated with a commercial halimeter. The sensor was also used to study the VSC levels in human subjects with healthy oral hygiene.
The level of VSC in mouth air of healthy subjects ranged between 0.2 and 0.4 μg L−1. The assay can be a potential diagnostic tool to evaluate any change in the VSC levels in mouth air due to different environmental factors.
The sensor is cost effective, sensitive and simple to use. It can be used for routine clinical evaluation of oral malodour and also as a self-test kit.
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GAS CHROMATOGRAPHY
Halicheck is a specialized Gas Chromatography test that measures the individual gases of bad breath. This machine is quite specialized and accurate that it can measure gas compounds as few as one part gas compound per billion parts of air.
Even low concentrations of bad breath gases, in particular Methyl Mercaptan play an important role in the pathogenesis of gum disease, as bad breath gases have been shown to be highly toxic to gingival tissue, and thus can accelerate a periodontal condition. 50
Disadvantages - Gas chromatographs are relatively
expensive Need personnel with special training to
operate them. The equipment is not portable and a
significant amount of time is needed to make each breath measurement.
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ADVANCES IN OCCLUSAL ANALYSIS
Articulating paper foils and ribbons have been used extensively in clinical practice as occlusal indicators.
Their clinical implementation requires the operator’s subjective interpretation of the markings to decide which contacts are acceptable, which are forceful, or which time premature is.
The marks’ appearance characteristics and strength of foil tug-back-descriptive of the occlusal load that created the mark.
Lastly, the presence of many similar-sized marks on neighboring teeth is purported to indicate equal occlusal contact intensity and evenness.
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Research on articulating paper mark size has revealed that the size of an articulating paper mark does not describe occlusal forces- Carey et al (2007)
As an alternative method to the operator’s subjective interpretation of articulating mark appearance, computerized occlusal analysis is available to the practitioner
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T-SCAN In 2006, a USB plug-in recording handle and
new generation of software were released as the T-Scan III occlusal analysis system.
The system displays a recorded occlusal “force movie,” which illustrates the various occlusal pressures with colors during playback. The darker colors represent low occlusal pressures and the brighter colors indicate higher occlusal pressures.
The T-Scan system is a valuable tool that aids in the diagnostic process of analyzing a patient’s bite to show what is and what is not functioning properly.
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KEY FEATURES
The ultra-thin, reusable sensor, shaped to fit the dental arch, inserts into the sensor handle, which connects into the USB port of your existing PC, making it easy to move from one operatory to another. Evaluating occlusal forces is as simple as having a patient bite down on the sensor while the computer analyzes and displays timing and force data in vivid, full-color 3-D or 2-D graphics. 56
KEY BENEFITS:
Improve clinical results Take back control, no longer rely on
patient feel Determine premature contacts Minimize destructive forces Provide instant documentation Use as a patient education tool to
enhance comfort and increase case acceptance
Save time by preventing remakes 57
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VIRTUAL DENTAL PATIENT
3-dimensional dental patient is assembled from the data scanned from the casts of a patient’s dentition.
This provides quantitative information that would aid in the assessment of his chewing function and in identifying the occlusal interferences.
Further, the sequential comparison of these occlusal contacts enables the dentist to identify the changes in the patient’s occlusion as time elapses.
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ADVANCES IN RADIOGRAPHIC DIAGNOSIS
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LIMITATIONS OF CONVENTIONAL RADIOGRAPHY
2D representation of 3D structures. superimposition of teeth and other anatomic
structures. Only interproximal alveolar bone levels can be
assessed with some level of certainty. Detection and quantitative assessment of 2-wall
and 3-wall defects remains a challenge Needs of substantial amount of mineral loss
(30%-50%) before bone resorption can be detected.
Misdirection of the central x-ray beam as well as exposure and processing errors.
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DIGITAL RADIOGRAPHY
Advantages Elimination of chemical processing Shorter exposure-to-display time Reduction in radiation exposure Integration with existing electronic office Quality image processing The software offers a variety of
measurement tools
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TWO TECHNOLOGIES CURRENTLY AVAILABLE
One uses solid-state detectors Other uses photostimulable phosphor
Solid-state detectors are based either on
charge-coupled device technology (CCD) or
complementary metal oxide semiconductor technology (CMOS)
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a) Kodak Insight F-speed film b) PSP plate c) CCD sensor d) CMOS sensor
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PHOTOSTIMULABLE PHOSPHOR(PSP)PLATES-
PSP plates resemble film with one of the sides lined with a PSP coating.
When interacting with x rays, PSP stores energy, which it then releases on stimulation by light of an appropriate wavelength.
The exposed plates are placed on a plate scanner and scanned by a laser beam
The radiographic image appears on a computer screen.
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DIGITAL SUBTRACTION RADIOGRAPHY
Based upon fact that when two images of the same object are registered and the image intensities of corresponding pixels are subtracted, a uniform difference image will be produced.
It cancels out the complex anatomic background against which this change occurs.
The conspicuousness of the change is greatly enhanced.
Subtraction images allow detection of mineral changes at little as 5%.
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Technique of Digital Subtraction Radiography(DSR)
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COMPUTED TOMOGRAPHY
Use a rotating fan beam to image one slice of the patient at the time, generally in an axial orientation.
Once the image volume has been generated, images slices can be reconstructed in various orientations through a process called multi-planar reformatting (MPR).
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Computed Tomography69
TUNED APERTURE COMPUTED TOMOGRAPHY(TACT)
Built on the basic principles of tomosynthesis: by shifting and combining a set of basic projections, arbitary slices through the object can be brought into focus.
The basic projections are conventional transmission
radiographs and each radiograph is taken from a different angle relative to the object and the receptor.
Each slice is a 2D representation of the object at a different location in the third dimension.
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LOCAL COMPUTED TOMOGRAPHY (LT)-
Local Computed Tomography is a form of Cone Beam Computed Tomography
Uses a small-field resolution detector to generate a limited high resolution three dimensional volume
Generates exquistic image detail in three dimensions while retaining the advantages of reduced patient dose and reduced cost.
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Local Computed Tomography72
OPTICAL COHERENCE TOMOGRAPHY
generates cross-sectional images of biological tissues using a near-infrared light sources.
The light is able to penetrate into the tissue without producing biologically harmful effects.
Differences in the reflection of the light are used to generate a signal that corresponds to the morphology and composition of the underlying tissues.
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ADVANTAGES
CAT eliminated superimpositions by mathematical recombination of multiple projections of a structure and provides across-sectional image, the elements of which are free of images of superimposed structures.
CAT permits the resolution of objects which differ only slightly in their attenuation of radiation. For example, the gray matter and white matter of the brain differ by 1% in physical density and by less than 1% in electron density, and yet CAT can distinguish the two tissues.
CAT data are acquired digitally and therefore offer greater flexibility in the processing, storage, transmission, analysis, and reformatting of images than does film-based imaging
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APPLICATIONS IN ORAL,REGION:
Anatomy Bone mass and bone mineralization:
quantitative CT- QCT is sensitive 3 to 4 times that of single-
photon absorptiometry and twice that of dual-photon absorptiometry,
has intermediate precision (2-5%, better than that of single-photon absorptiometry but not as good as that of dual-photon methods),
and variable accuracy of 5-20% (spanning the range of other available methods).
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Dental implant treatment planning: 2 dimensional reformats of CT scans of the
maxillary or mandibular alveolar process have been used for treatment planning of dental implants.
"Panoramic" 2-D reconstructions of the length of the alveolar ridge and cross-sectional reconstructions of the buccolingual and supero-inferior dimensions of the ridge may be obtained through proprietary software packages, by eliminating superimpositions and distortion.
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Indications of CAT for dental implantology includes:
posterior maxillary or mandibular sites where conventional radiography shows inadequate bone,
anterior maxillary sites when multiple implants are planned,
cases in which complete maxillary or mandibular subperiosteal implants are planned, and
cases in which an evaluation of buccolingual ridge dimensions is required.
Periodontal Diagnosis
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DENTA SCAN
DentaScan is a software program developed to automatically reformat the oblique cross-sectional images.
It permits visualization of the mandible and maxilla in three planes: axial, oblique sagittal (cross-sectional), and panoramic
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It allows internal structures such as the inferior alveolar canal, incisive canal, and maxillary sinuses to be seen in cross section.
Precise millimeter measurements of the height and width of the alveolar ridge can be obtained without the distortion typically seen with standard panoramic views.
Buccolingual atrophy and contour irregularities are readily assessed, and the optimal site for implantation can be determined.
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3 roles in managing patients for dental implant :
It preoperatively identifies patients who have insufficient bone for implantation, obviating the need to contend with this sometimes unanticipated condition during surgery. Alternative treatment plans can be made in advance.
It identifies the optimum site for implantation by locating the exact position of the inferior alveolar canal and maxillary sinuses and the area of maximum bone height, width, and density.
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It identifies implant sites in patients who, based on standard radiographs, were once considered to be inoperable because of insufficient bone.
In addition to its use in implant surgery, DentaScan is proving to have more expanded applications. It not only identifies ideal implant sites but also depicts various types of pathology such as periapical and periodontal disease, sinus disease, tumors, root resorption, and foreign bodies. 81
COMPUTER-ASSISTED DENSITOMETRIC IMAGE ANALYSIS (CADIA)
A video camera measures the light transmitted through the radiograph, and the signals from the camera are converted into gray-scale images.
The camera is interfaced with an image processor and a computer that allows the storage and mathematic manipulation of the images.
Shows higher sensitivity, reproducibility and accuracy.
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MAGNETIC RESONANCE IMAGING
MR images are obtained measuring changes in low frequency radio signals in the magnetic field.
The resulting data can be used to create images of the structures examined or chemical profile of the tissues.
This technology gives better soft tissue images than CT and the patient is not exposed to radiation.
MR imaging is mainly used in the study of TMJ and the soft tissue lesion of gingiva and other oral structures. 83
ADVANCES IN MICROBIOLOGIC ANALYSIS
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ADVANTAGES OF MICROBIAL TESTING
Determination of bacteria associated with disease in certain individuals ⁄ sites
Aids in selection of the most appropriate antibiotics
Avoidance of arbitrary antibiotic prescription and possible increase in bacterial resistance
Determination of bacterial elimination ⁄ reduction in number after treatment to correlate with disease progression ⁄ resolution
Detection of the emergence of resistant strains post-therapy
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TECHNIQUES FOR DETECTION OF MICROBES
Culturing Phase contrast microscopy Immunologic assays DNA probes Enzyme tests Molecular microbial diagnosis
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CULTURING Involve growing the microorganisms on defined
media, followed by identification based on phenotypic and biochemical criteria, differential staining methods, metabolic end-product analysis and cell membrane composition.
Advantages identify microbes obtain relative and absolute counts of the
cultured species characterize new species determine antibiotic susceptibility of oral
microbes and pathogenicity of individual species.
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Shortcomings Can only grow viable bacteria Treponema sp, Tf are difficult to culture Sensitivity low Detection limit- 103-104 bacterial cells Requires specific laboratory equipment Time consuming Expensive
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PHASE AND DARK-FIELD MICROSCOPY
For motile forms- spirochetes Main putative periodontopathogens,
including Aac, Pg, Bf, Ec, and Eubacterium species, are nonmotile, and therefore this technique is unable to identify these species.
It is also unable to differentiate among the various species of Treponema.
An unlikely candidate as a diagnostic test of destructive periodontal diseases
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IMMUNOLOGIC ASSAYS
Direct and Indirect Immunofluorescent microscopy
Latex agglutination
Flow cytometry
ELISA
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ENZYMATIC METHODS OF BACTERIAL IDENTIFICATION- BANA TEST
B. forsythus, P. gingivalis, T. denticola, and Capnocytophaga species - 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-DL-arginine-2-naphthylamide (BANA).
When the hydrolysis takes place, it releases the chromophore ß-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).
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Loesche et al (1986) 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 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 (1995) used the BANA test as a risk indicator for periodontal attachment loss.
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Limitations: It may be positive at clinically healthy
sites and remains to be proven whether this test can detect sites undergoing periodontal destruction.
Since it only detects a very limited number of pathogens, its negative result does not rule out the presence of other important periodontal pathogens.
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MOLECULAR MICROBIAL DIAGNOSIS
DNA Probes Polymerase chain reaction (PCR) based
methodso Single target PCRo Multiplex PCRo Quantitative PCR
DNA-DNA hybridization methodso In situ hybridizationo Checkerboard hybridizationo 16S ribosomal RNA based microarrays
Sequencing methodso Pyrosequencingo Real-time single-molecule DNA sequencingo Nanopore-based sequencing
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DNA PROBES
• DNA obtained from pure cultures are enzyme-digested,
• Specific fragments of single strands are then radiolabelled and serve as a "DNA library" for future tests.
• Plaque samples submitted for assay are enzymatically digested, and their fragmented single strands are attached by chemical treatment to a nitrocellulose filter.
• Fragments are then exposed to labeled single strands from the DNA library.
• Attachment of complementary strands to each other
• Washing away unlinked fragments.
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Linked strands attached to the filter are exposed to auto radiographic plates to determine the extent hybridization occurred
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COMMERCIAL CHAIRSIDE DIAGNOSTIC KITS
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Commercially available kit Bacteria identified
DMDx Patho Tek (DNA) A.comitans,P.gingivalis, P.intermedia,
T.forsythus,C.rectus, T.denticola,
F.nucletum.
Meridol DNA proe test 3/8 A.comitans, P.gingivalis, P.intermedia,
T.denticola, T.forsythus.
Microdent Test( DNA) A.comitans, P.gingivalis, P.intermedia,
T.denticola, T.forsythus.
Perio Bac Test(DNA) A.comitans, P.gingivalis, P.intermedia,
T.denticola, T.forsythus
Omnigene and BTD A.comitans, P.gingivalis, P.intermedia
PCR-BASED METHODS- DEVELOPED IN 1983 BY KARY MULLIS
pcrSingle target PCR applications
For the detection of putative pathogenic species typically associated with periodontal disease, such as P.gingivalis, T. forsythus, T.denticola, A.actinomycetemcomitans.
Species specific or phylotype-specific PCR primers designed and subsequently used in highly stringent, individual PCR reactions.
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MULTIPLEX PCR
is an expansion of single target PCR methodology
more than one pair of species specific primers is
used in a single PCR assay and that permits
multiple species to be detected simultaneously.
used to detect A. actinomycetemcomitans, T.
forsythia and P. gingivalis at the same time.
detection limits of 10–100 cells per PCR reaction.
The MicroDent Test is a commercially available
method using multiplex PCR. 99
REAL-TIME PCR
Real Time PCR is characterized by the point in time during cycling when amplification of the PCR product of interest is first detected rather than the amount of the PCR product of interest which is accumulated at the end-point after PCR which contained a large number of cycles.
Real Time PCR does this by monitoring the amount of fluorescence emitted during the PCR reaction, and this acts as an indicator of the amount of PCR amplification that occurs during each PCR cycle.
Thus, in newer Real Time PCR machines, one can visually see the progress of the reaction in "real time".
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Detects and quantify A. actinomycetemcomitans, P. gingivalis, Prevotella intermedia, the tetQ gene and total bacteria, in clinical samples.
MyPerioPath from Oral DNA labs is a commercially available service that utilizes TaqMan PCR to determine the presence and the microbial profile of 13 putative periodontal pathogens from oral specimens provided by clinicians.
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DNA-DNA HYBRIDIZATION METHODSFLUORESCENCE IN SITU HYBRIDIZATION- FISH
FISH or more specifically whole-cell hybridization- quantify, determine the spatial configuration and demonstrate the morphology of individual bacterial cells in dental plaque.
visualized using fluorescence microscopy or confocal fluorescence microscopy.
Aac, Pg, Actinomyces spp. and Streptococcus spp., and phylotypes known only from 16S ribosomal RNA sequence analysis, have been detected
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CHECKERBOARD HYBRIDIZATION
This method was introduced in 1994, enabled the hybridization of 45 DNA samples against 30 DNA probes (i.e. up to 1,350 simultaneous hybridizations) on a single support membrane.
Socransky et al developed this technique for the detection to evaluate levels of 40 bacterial species often found in the oral cavity. It is based upon similar principle of DNA probes but the assay uses whole genomic, digoxigenin –labelled DNA probes and facilitates rapid processing of large numbers of plaque samples with multiple hybridization for up to 40 oral species in as single test.
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Hybridization signals are typically detected using chemifluorescence procedures.
The reason it is called “checkerboard” is that the genome or ribosomal RNA probes are hybridized at right-angles to the DNA of multiple oral samples, and processed images of the hybridizations look like a checkerboard.
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OLIGONUCLEOTIDE MICROARRAY TECHNOLOGY The human oral microbe identification
microarray was developed in order to examine the complex oral microbial diversity in a single hybridization reaction on glass slides.
16S ribosomal RNA genes are PCR-amplified from DNA isolated from clinical samples and labelled.
Labeled amplicons are hybridized to the 16S ribosomal RNA-based, reverse-capture oligonucleotide probes printed on aldehyde-coated glass slides.
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To analyze data from human oral microbe identification microarrays, individual signals are translated to a ‘bar code’ format and are normalized by comparing individual signal intensities with the average of signals from universal probes.
The bands correspond to presence or absence, and band intensities correspond to 1+, 2+, 3+, 4+, or 5+ thus more intense bands reflect higher proportions.
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Human oral microbe identification microarrays have been used to compare the subgingival microbiota of patients with refractory periodontitis, successfully treated periodontitis and a periodontally healthy oral environment.
Reverse-capture, 16S ribosomal RNA-based microarray, ParoCheck DNA chip targets 20 oral bacterial species.
High-density 16S ribosomal RNA-based microarray, Phylochip can detect up to 32,000 16S ribosomal RNA phylotypes.
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PYROSEQUENCING
A sequence of enzyme-triggered reactions occurs, which ultimately results in the production of a luminescence signal.
Because the order in which the deoxyribonucleotide triphosphates are added is known and a charge-coupled device camera records the intensity of the luminescence bursts, the template sequence and its quantity can be reconstructed.
Pyrosequencing has been used to identify mycobacteria, clinically relevant yeasts and pathogenic Candida species.
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NEXT-GENERATION TECHNOLOGIES AND CLINICAL IMPLICATIONS
There are several advantages of characterizing genes by sequencing, rather than by using microarray hybridization.
Specifically, sequencing does not require knowledge of the targets to be hybridized, gene sequencing provides a “digital” rather than an “analog” signal, and once a gene has been sequenced, putative identification of the gene can be rendered by comparing the sequence to reference genes in DNA databases.
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3 1st next-generation sequencing devices are
Roche ⁄ 454 (Roche 454 Life Sciences, Branford,CT)
Illumina ⁄ Solexa (Illumina, San Diego, CA)
Applied Biosystems ⁄ SOLiD (Life Technologies, Carlsbad, CA).
The first-generation systems rely on DNA amplification and therefore do not permit single molecule detection.
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Second next-generation sequencing system: Pacific Biosystem system
Third next-generation sequencing system: Oxford Nanopore Technology NABsys Second- and third generation next-
generation sequencing systems can detect single molecules.
They are based on nanopore technology, they do not require polymerase and they are not yet commercially available.
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ADVANCES IN CHARACTERIZINGTHE HOST RESPON SE- DIAGNOSTIC BIOMARKERS
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REQUISITES FOR A PERIODONTAL DIAGNOSTIC MARKER
It should be able to distinguish periodontally healthy sites from those sites affected by gingivitis and periodontitis.
It should be able to differentiate between progressive and non-progressive lesions.
It should indicate the presence of a disease process before extensive clinical damage has occurred.
It should have high specificity and sensitivity. It should have ease of use either as a chair
side procedure, a home screening device or test.
It should be cost-effective.
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SOURCES
Potential sample sources include saliva, gingival crevicular fluid (GCF), gingival crevicular cells, blood serum, blood cells, and urine.
Analysis of urine only for differential diagnosis of tooth loss related to hypophosphatasia in young children, in whom the presence of phosphoethanolamine in urine is diagnostic of the disease.
Most efforts to date have been based on the use of components of GCF and, to a lesser extent, saliva and blood.
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POTENTIAL MARKER SOURCES OF PERIODONTAL DISEASE ACTIVITY IN GCF
Host derived enzymes and their inhibitors- released from dead and dying cells of the periodontium; PMNs and other inflammatory cells, epithelial, and connective tissue cells at affected sites.
Inflammatory mediators and host response modifiers- TNF- α, IL- 1α, IL-1ß, IL-6, and IL-8.
Tissue breakdown products- hydroxyproline from collagen breakdown and glycosaminoglycans from matrix degradation.
Other bone and connective tissue proteins, including osteocalcin and type 1 collagen peptides, have been correlated with the progression of alveolar bone loss. ICTP – Specific marker of bone loss.
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GROUPS OF POTENTIAL MARKER SOURCES OF PERIODONTAL DISESE ACTIVITY IN GCF
Microbial Plaque Endotoxins (lipopolysaccharide)EnzymesMetabolic end products
Host Cells Leucocytic enzymesLactoferrinLysozyme
Host Tissue CollagensProteoglycansMatrix proteins
Host Factors: Immune Response ImmunoglobulinsComplementEicosanoidsCytokines
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HOST-DERIVED ENZYMES AND THEIR INHIBITORS IN GCF
Aspartate aminotransferase
Alkaline phosphatase
Acid phosphatase
Β-Glucouronidase
Elastase
Elastase inhibitors1. α₂-Macroglobulin2. α₁-Proteinase inhibitor
Cathepsins1. Cysteine proteinases (B,H,L)2. Serine proteinase (G)3. Cathepsin D
Trypsin-like enzymes
Immunoglobulin-degrading enzymes
Glycosidases
Dipeptidyl peptidases
Non specific neutral proteinases
Collagenases
Gelatinases
Tissue inhibitor of MMP-1
Stromyelysins
Myeloperoxidases
Lactate dehydrogenase
Arylsulfatase
Creatinine kinase
β-N-acetyl-hexosaminidase 117
INFLAMMATORY MEDIATORS AND HOST RESPONSE MODIFIERS IN GCF
Cytokines1. Interleukin-1α2. Interleukin-1β3. Interleukin-1ra4. Interleukin-25. Interleukin-66. Interleukin-87. Tumor necrosis factor α8. Interferon α
RANTES (chemoattractant and activator of macrophages and lymphocytes)
Prostaglandin E₂ Leukotriene B₄ Acute – phase proteins
Autoantibodies- Anti-desmosomal antibody Antibacterial antibodies
Plasminogen activator (PA) PA inhibitor-2 (PAI-2) Substance P Vasoactive intestinal peptide Neurokinin A Platelet-Activating Factor CD14 Cystatins Calgranulin A (MRP-8)
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TISSUE BREAKDOWN IN GCF
Glycosaminoglycans1. Hyaluronic acid2. Chondroitin – 4 – sulfate 3. Chondroitin – 6 – sulfate 4. Dermatan sulfate
Hydroxyproline Fibronectin fragments Connective tissue and bone proteins1. Osteonectin2. Osteocalcin3. Type I collagen peptides4. Osteopontin
Laminin Calprotectin Hemoglobin β-chain peptides Pyridinoline crosslinks (ICTP) 119
BONE FORMATION MARKERS
1. Type I procollagen propeptide – proliferation C-terminal propeptide fragment (PICP) N-terminal propeptide fragment (PINP)
1. Alkaline phosphatase – matrix formation Total alkaline phosphatase (Al-p) Bone alkaline phosphatase (Bal-p)
1. Osteocalcin, bone Gla protein – mineralization (BGP) C-terminal fragment Mid-portion Intact
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BONE RESORPTION MARKERS
1. Pyridinium cross-link Urine pyridinoline (PYP), deoxypyridinoline (DPD), HPLC method Urine free deoxypyridinoline (fDPD)
2. Pyridinium cross-link collagen peptide fragment Serum C-terminal telopeptide (ICTP) Urine C-terminal telopeptide (CTx, crosslaps®) Urine N-terminal telopeptide (NTx, osteomark®)
3. Tartrate-resistant acid phosphatase
4. Galactosyl hydroxylysine (GHYL)
5. Hydroxyproline
6. N-terminal osteocalcin fragment
7. Glycosaminoglycans (GAGs) 121
COMMERCIAL CHAIRSIDE DIAGNOSTIC KITS
COMMERCIAL EXAMPLE
TEST SITE
PERIOGARD (COLGATE) Detection of Aspartate Amino Transferase (AST) in GCF
GCF
PROGNOSTIK (DENTSPLY)
Serine, Proteinases and Elastase in GCF samples
GCF
PERIOCHECK (ACTECH) Proteinases like Collagenase GCF
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POSSIBLE SALIVARY MARKERS FOR PERIODONTAL DIAGNOSIS
Enzymes Immunoglobulins Proteins Host cells Ions Hormones Bacteria Volatile compounds
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Enzymes found in whole saliva originate from three main sources:
i) the actual salivary secretions ii) the GCF, stemming from PMNs and
tissue degradationiii) disposed bacterial cells from dental
biofilms and mucosal surfaces.
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ELECTROCHEMICAL SENSOR FOR MULTIPLEX BIOMARKERS DETECTION
Electrochemical sensor (Fang et al. 2009) is used for oral cancer detection based on the simultaneous detection of two salivary biomarkers: IL-8 mRNA and IL-8 protein.
Multiplex assay of these 2 biomarkers directly from 28 cancer and 28 matched control saliva samples shows significant clinical discrimination.
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From the receiver operating characteristic analysis, the EC sensor yielded 90% sensitivity and specificity for both IL-8 mRNA and IL-8 protein.
The good correlation between EC sensor and PCR/ELISA suggests that the EC sensor is promising for clinical diagnostics.
The EC sensors are 16 integrated gold electrode arrays.
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Probes for mRNA and protein are precoated onto different electrodes.
A sandwich assay technique is carried out for both salivary mRNA and protein.
This multiplexing assay of mRNA and protein simultaneously has the advantage of high accuracy.
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ADVANTAGES :
The traditional PCR and ELISA technologies have difficulties in obtaining both sensitivity and specificity under the same condition, which is ruled out with EC.
With EC, both hybridization and protein binding are finished on the same chip within minutes (200 min. approx.).
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RAPID POINT-OF-CARE DIAGNOSTICS FOR PERIODONTAL DISEASE
The portable, easy to use diagnostic tools will allow patients to be screened for periodontal
disease in settings other than the dental practice will also enable screening of large populations
‘Lab-on-a-chip’ and microfluidic devices have the potential to manage complex oral fluids such as saliva and GCF, and to provide a determination of a patient’s periodontal disease risk profile, current disease activity, and response to therapeutic interventions.
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Strategy for oral fluid sampling and analysis with rapid point-of-care or lab-on-chip device for the generation of
periodontal disease biomarker report130
ADVANCES IN GENETIC DIAGNOSIS
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Commercially available kits
Patient susceptibility test- Interleukin Genetics
Genotype PRT- Hain ParoGen Test- IAI
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POTENTIAL TYPES OF THE PATIENT’S AND THE BENEFITS THAT MAY BE DERIVED FROM THE DECISION TO DETERMINE PATIENT’S GENOTYPE
Patients with early signs of periodontal disease.
Patients who are resistant to accepting treatment recommendation.
New periodontal patients, with family history of periodontal problems .
Patients not responding to routine periodontal procedures.
Biologic family members of genotype positive patients
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PST GENETIC TEST First genetic test that analyzes two IL-1
genes for variations that identify an individual’s predisposition for over expression of inflammation and risk for periodontal disease.
IL-1 genetic susceptibility may not initiate or cause the disease but rather may lead to earlier or more severe disease.
This test is not intended to and does not diagnose a specific disease.
It can be used to differentiate certain IL1 genotypes associated with varying inflammatory responses to identify individuals at risk for severe periodontal disease even before age 60.
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TREATMENT DECISIONS INFLUENCED BY GENETIC TESTING
The finding that a patient is genotype- positive does not indicate that the patient will develop severe periodontitis.
Papapanou et al (2001) reported that among healthy patients and patients with periodontitis, the percentage of genotype-positive patients was 41.7% and 45.2% respectively.
In the study by Kornman and colleagues (1997)- 38 %of the patients with moderate periodontitis were genotype-positive. In addition, 33% of patients with severe periodontitis were genotype-negative.
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Thus, detection of a positive genotype is not sufficient to initiate therapy, and the finding of a negative genotype is inadequate to conclude that the patient will not develop severe periodontitis.
Even if the presence of specific genetic polymorphisms associated with an increased risk of developing periodontitis is identified, periodontitis will not develop if the bacterial pathogens do not overwhelm the host response. 137
If a genetic test is used to identify people at increased risk of developing a disease, clinicians must be aware that the age of the patient, the part of the dentition to become affected and the percentage of genotype-positive patients who will manifest severe periodontitis all are unknown.
No intervention studies have been performed based on genotype determinations.
Thus, we do not know the extent to which treatment needs to be altered among patients identified at risk in order to reduce the incidence of periodontitis.
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ORAL RINSE TEST TO DIAGNOSE PERIODONTAL DISEASE
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ORAL RINSE TEST
a simple and rapid method for the quantification of oral neutrophil levels in saliva.
is useful for examining the periodontal breakdown and the effectiveness of periodontal therapy.
utilizes a 30 second oral rinse that is collected from the patient.
A biochemical agent is added that results in a colour change that reports the number of oral neutrophil present.
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CONCLUSION
In most of the cases traditional diagnostic tests are sufficient to diagnose the specific disease but some cases which are refractory and do not respond well to the planned treatment modalities may require specialized/ advanced diagnostic tests.
Gathering more information simply because we have the capability to do so does not make any sense and nor it automatically translates into better treatment for improved patient out come unless they are properly understood and applied by the clinician.
There is still lack of a proven diagnostic test that has demonstrated high predictive value for disease progression, has proven impact on disease incidence and prevalence, and is simple, safe and cost effective.
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THANKS
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