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 33
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.
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
Limitations of conventional techniques Conventional diagnostic techniques do not Ø reliably differentiate sites with ongoing periodontal destruction and inactive sites Ø provide information on the patient’s susceptibility to disease Ø differentiate disease is progressing or in remission
Advances in clinical diagnosis
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
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 1010
Limitation Precision Range Probing Force Applicability Reach Angulation Readout Security Conventional 1mm 12mm Non-standardized Noninvasive Easy to access Subjective Voice dictation and recording Easily sterilized NIDCR Criteria 0.1mm 10mm Constant Noninvasive Easy to access Guidance System Direct electronic reading Complete sterilization
5/4/12 Generations of Periodontal probes Type Characteristics First Generation: conventional probe ;manualUsual clinical instrument : a thin tapering tine probes. marked to be read in mm. Second generation: constant force probes.As above, but with a spring or electronic cutPressure sensitive probe. out when the appropriate fore is reached. Force (given by Vander Velden 1978) 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 These are aimed at recording sequential probe positions along a gingival sulcus non-invasive threeThese will add ultrasound or another device to a fourth generation probe.Identifying attachment level with and penetrating it . 1212
manual probes like Ø William’s Periodontal probe Ø UNC 15 probe Ø CPITN Probe Ø Michigan Probe etc.
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.
Second Generation Probes (pressure sensitive)
- to reduce measurement variation by standardizing probing force. Probes available : Gabathuler probe True Pressure-sensitive probe (Vivacare) Prockprobe PDT Sensor Probe
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
In 1977, Armitage designed a pressuresensitive 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 pressuresensitive probe with a cylinder and piston connected to an air-pressure system. Subsequently, it was modified with a displacement transducer for electronic pocketdepth reading. 1717
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 1818
Third Generation Probes (Automated/computer linked electronic constant pressure)
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).
by Gibbs et al (1988). Advantage : Ø Constant probing force Ø Precise electronic measurements Ø Computerised data capturing Ø Complete sterilization
lack tactile sensitivity
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. 2121
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.
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 0.5mm nickel-titanium wire that is extended under air pressure.
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
Fourth Generation Probes (Ultrasonic )
a very narrow beam of highfrequency (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
detects much smaller increments of anatomic change Ø earlier detection of tissue breakdown Ø additional histological information, such as tissue thickness and inflammation Ø permits earlier diagnosis and intervention Ø Handpiece design is ergonomically modified 2727
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
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 2929 delivering sufficient signal strength and depth
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
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 3131
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
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, wellbalanced 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. 3333 As with many automated probes, there is
Automated calculus-detection technologies
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
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 gramnegative 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 3636
Diagnosing tooth mobility
System (Schulte et al.
1992) Ø The use of the Periotest, a noninvasive, electronic device that provides an objective measurement of the reaction of the periodontium to a defined impact load.
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)
Laser Vibrometer Method
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.
Resonance Frequency Analysis
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 transducerimplant unit is calculated.
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. 4242
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.
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 recalibration 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 4444
5/4/12 ADVANCES IN DIAGNOSIS OF ORAL MALODOR
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 4545
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.
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.
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
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. 5050
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.
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 to5353
ADVANCES IN OCCLUSAL 5/4/12 ANALYSIS
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
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. 5555
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. 5656
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
Virtual dental patieNT 3-dimensional dental patient is assembled from the data scanned from the casts of a patient’s dentition. ØThis provides quantitative information that would aid in the assessment of his chewing function and in identifying the occlusal interferences. ØFurther, the sequential comparison of these occlusal contacts enables the dentist to identify the changes in the patient’s occlusion as time elapses. 5959
Advances in radiographic diagnosis
Limitations of conventional radiography
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
Elimination of chemical processing Ø Shorter exposure-to-display time Ø Reduction in radiation exposure Ø Integration with existing electronic office Ø Quality image processing Ø The software offers a variety of measurement tools
Two technologies currently available
One uses solid-state detectors Ø Other uses photostimulable phosphor
Ø Solid-state ü ü
detectors are based either
on charge-coupled device technology (CCD) or complementary metal oxide semiconductor technology (CMOS)
a) Kodak Insight F-speed film b) PSP plate c) CCD sensor d) CMOS sensor
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 6565
Digital Subtraction Radiography
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.
Technique of Digital Subtraction Radiography(DSR)
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).
Tuned Aperture Computed Tomography(TACT)
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.
basic projections are conventional transmission radiographs and each radiograph is taken from a different angle relative7070
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.
Local Computed Tomography
Optical Coherence Tomography
cross-sectional images of biological tissues using a near-infrared light sources. 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 7373
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.
Applications IN ORAL,REGION:
Anatomy Bone mass and bone mineralization: quantitative CT QCT is sensitive 3 to 4 times that of singlephoton 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).
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 crosssectional reconstructions of the buccolingual and supero-inferior dimensions of the ridge may be obtained through proprietary software packages, by eliminating
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 7777 ridge dimensions is required.
is a software program developed to automatically reformat the oblique crosssectional images. permits visualization of the mandible and maxilla in three planes: axial, oblique sagittal (cross-sectional), and panoramic
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.
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 8080
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. 8181
video camera measures the light transmitted through the radiograph, and the signals from the camera are converted into gray-scale images. camera is interfaced with an image processor and a computer that allows the storage and mathematic manipulation of the images.
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. 8383
ADVANCES IN MICROBIOLOGIC ANALYSIS
Advantages of Microbial testing
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
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 Ø identify microbes Ø obtain relative and absolute counts 8787
Can only grow viable bacteria Ø Treponema sp, Tf are difficult to culture Ø Sensitivity low Ø Detection limit- 103-104 bacterial cells Ø Requires specific laboratory equipment Ø Time consuming Ø Expensive
Phase and Dark-Field Microscopy
For motile forms- spirochetes Ø Main putative periodontopathogens, including Aac, Pg, Bf, Ec, and Eubacterium species, are nonmotile, and therefore this technique is unable to identify these species. Ø It is also unable to differentiate among the various species of Treponema. Ø An unlikely candidate as a diagnostic test of destructive periodontal diseases
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-DLarginine-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
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 9292
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.
Molecular microbial diagnosis
DNA Probes ü Polymerase chain reaction (PCR) based methods Single target PCR Multiplex PCR Quantitative PCR ü DNA-DNA hybridization methods In situ hybridization Checkerboard hybridization 16S ribosomal RNA based microarrays ü Sequencing methods Pyrosequencing Real-time single-molecule DNA sequencing Nanopore-based sequencing
o o o
o o o
o o o
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
Linked strands attached to the filter are exposed to auto radiographic plates to determine the extent hybridization occurred
PCR-based methods- Developed in 1983 by Kary
pcr Single target PCR applications
the detection of putative pathogenic species typically associated with periodontal disease, such as P.gingivalis, T. forsythus, T.denticola, A.actinomycetemcomitans. specific or phylotype-specific 9898
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.
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.
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.
DNA-DNA HYBRIDIZATION 5/4/12 METHODS
Fluorescence 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 102
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. et al developed this technique for the detection to evaluate levels of 40 bacterial species often found in the oral cavity. It is based
signals are typically detected using chemifluorescence procedures. 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.
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 PCRamplified from DNA isolated from clinical samples and labelled. Labeled amplicons are hybridized to the 16S ribosomal RNA-based, reverse-capture 105 oligonucleotide probes printed on
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. bands correspond to presence or absence, and band intensities correspond to 1+, 2+, 3+, 4+, or 5+
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. 16S ribosomal RNAbased microarray, ParoCheck DNA chip targets 20 oral bacterial species.
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.
5/4/12 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.
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 110
Second next-generation sequencing system: Pacific Biosystem system Third next-generation sequencing system: Oxford Nanopore Technology NABsys Second- and third generation nextgeneration sequencing systems can detect single molecules. They are based on nanopore technology, they do not require polymerase and they are not yet 111
ADVANCES IN CHARACTERIZING Click to edit Master subtitle style THE HOST RESPON SEDiagnostic biomarkers
Requisites for a periodontal 5/4/12 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 113 test.
sample sources include saliva, gingival crevicular fluid (GCF), gingival crevicular cells, blood serum, blood cells, and urine. of urine only for differential diagnosis of tooth loss related to hypophosphatasia in young children, in whom the presence of phosphoethanolamine in urine is
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, IL115
GROUPS OF POTENTIAL MARKER SOURCES OF PERIODONTAL DISESE ACTIVITY IN GCF Microbial Plaque Endotoxins (lipopolysaccharide) Enzymes Metabolic end products Leucocytic enzymes Lactoferrin Lysozyme Collagens Proteoglycans Matrix proteins Immunoglobulins Complement Eicosanoids Cytokines
Host Factors: Immune Response
HOST-DERIVED ENZYMES AND THEIR INHIBITORS IN GCF Trypsin-like enzymes
• • •
Glycosaminoglycans Hyaluronic acid Chondroitin – 4 – sulfate Chondroitin – 6 – sulfate Dermatan sulfate Hydroxyproline Fibronectin fragments Connective tissue and bone proteins Osteonectin Osteocalcin Type I collagen peptides Osteopontin Laminin Calprotectin Hemoglobin β-chain peptides Pyridinoline crosslinks (ICTP)
BONE FORMATION MARKERS
Type I procollagen propeptide – proliferation • C-terminal propeptide fragment (PICP) • N-terminal propeptide fragment (PINP) Alkaline phosphatase – matrix formation Total alkaline phosphatase (Al-p) Bone alkaline phosphatase (Bal-p) Osteocalcin, bone Gla protein – mineralization (BGP) C-terminal fragment Mid-portion Intact
found in whole saliva originate from three main sources: i) the actual salivary secretions ii) the GCF, stemming from PMNs and tissue degradation iii) disposed bacterial cells from dental biofilms and mucosal surfaces.
ELECTROCHEMICAL SENSOR FOR 5/4/12 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.
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.
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.
traditional PCR and ELISA technologies have difficulties in obtaining both sensitivity and specificity under the same condition, which is ruled out with EC. EC, both hybridization and protein binding are finished on the same chip within minutes (200 min. approx.).
Rapid point-of-care diagnostics for periodontal disease
The portable, easy to use diagnostic tools will
patients to be screened for periodontal disease in settings other
Strategy for oral fluid sampling and analysis with rapid point-of-care or lab-on-chip device for the generation of periodontal disease biomarker report
potential types of the patient’s and the benefits 5/4/12 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
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 134
5/4/12 TREATMENT DECISIONS INFLUENCED BY GENETIC TESTING
The finding that a patient is genotypepositive 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 genotypepositive patients was 41.7% and 45.2% respectively. In the study by Kornman and colleagues (1997)38 %of the patients with moderate periodontitis were genotypepositive. In addition, 33% of patients with 136
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 genotypepositive 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
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ORAL RINSE TEST TO DIAGNOSE PERIODONTAL disease
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.