GOOD MORNING 1Dr Ashish U. Bisane
Advanced Diagnostic
Aids
2By: Dr Ashish U. Bisane
Introduction
• Periodontal disease is considered a site
specific disease characterized by local
inflammatory reaction to b...
Gingival Bleeding
• Gingival Inflammation is diagnosed based upon:
1. Gingival Bleeding on Probing (Objective sign)
2. Gin...
• To test for bleeding
after probing, the probe
is carefully introduced
to the bottom of the
pocket and gently
moved later...
• Subjects who refrain from normal oral hygiene procedures have a resultant increase
in plaque accumulation and demonstrat...
• Drawbacks:
1. Healthy sites may bleed on probing as any force
greater than 0.25N evokes bleeding even in an intact
perio...
(Sub)Gingival Temperature
• Kung et al, 1990; demonstrated that the
subgingival temperature at diseased sites is
increased...
Perio-Temp (Thermal
Periodontal Probing System)
• Perio-Temp Probe (Abiodent)
• Enables the calculation of temperature
dif...
Perio-Temp
11Dr Ashish U. Bisane
• Haffajee et al used this probe to assess its predictability in identifying loss of
attachment, concluding that sites wit...
• The reason why temperature increases with probing
depth is unclear. A possible explanation is an increase
in cellular an...
Periodontal Probing
• Currently the “Gold Standard” for recording changes in
periodontal status in longitudinal measuremen...
Drawbacks in manual
probing:
1. Use of the periodontal probe - many problems in terms of sensitivity and
reproducibility o...
Probing Force
• Penetration of probe has been positively
correlated with probing force. (Mombelli.A,
1986)
• With forces u...
Classification
• First generation probes/ conventional
• Second generation probes/pressure sensitive
• Third generation pr...
18Dr Ashish U. Bisane
National Institute of Dental and Craniofacial Research (NIDCR) criteria
for overcoming limitations of conventional periodo...
Florida Probe System
• Gibbs et al - developed Florida Probe System (Florida Probe
Corporation)
• Parts of Florida Probe S...
Advantages
1. Constant probing force
2. Precise electronic measurement
3. Computer storage of data
Disadvantages
1. Lack o...
22Dr Ashish U. Bisane
23Dr Ashish U. Bisane
Florida Probe System to
Record CAL
• CALs are recorded relative to a fixed reference
point, such as the occlusal surfaces ...
25Dr Ashish U. Bisane
Florida PASHA Probe
• Modification of Florida probe to detect CEJ.
• Has a Modified Sleeve, which includes a
prominent 0.1...
Toronto Automated Probe
• The uses the occlusal-incisal surface to measure
relative clinical attachment levels.
(Mcculloch...
Interprobe & Perioprobe
• An electronic probe using an optical encoder transduction element -
evaluated by Goodson and Kon...
Probes and the examination of
patients considered for implants
• Examination of patients -
considered for implants include...
30Dr Ashish U. Bisane
31Dr Ashish U. Bisane
Drawbacks of Conventional
Radiography
• Conventional radiographs are very specific but lack
sensitivity.
• Considerable am...
Solutions for Standardization
of Radiographic Techniques
• Radiographs should be obtained in a constant
reproducible plane...
Digital Radiography
• Digital radiography enables the use of computerized
images - stored, manipulated, and corrected for ...
Types of Digital Intraoral Radiographic
Systems (Parks.E.T. 2002)
• First system
1. Charge – Coupled Devices (CCDs)
2. Com...
• Second System: uses Photostimulable Phosphor (PSP) plates as
detectors.
• PSP plates resemble film with one of the sides...
The Digital Advantage
1. Speed of image capture and display
2. Low x-ray exposure
3. Ability to manipulate the image and m...
• Once captured and displayed, computer software can be
used to enhance the digital image and increase its diagnostic
effi...
Subtraction Radiography
• This technique requires a paralleling technique to obtain a
standardized geometry and accurate s...
Advantages:
High correlation between alveolar bone loss
and CAL changes
Increased detection of small osseous lesion
Bot...
• Studies using this technique have shown:
1. A higher degree of correlation between changes
in alveolar bone determined b...
Serial radiographs
converted to digital images
superimposed
composite image
Quantitative changes 43Dr Ashish U. Bisane
• The disadvantage is the need to be almost at
identical projection alignment during the
exposure of sequential radiograph...
Diagnostic/Digital
Subtraction Radiography (DSR)
• Combines the use of a positioning device during
film exposure with a sp...
Computer Assisted Densitometric
Image Analysis System (CADIA)
• A video camera measures the light transmitted through
the ...
Advances in
Microbiological
Analysis
47Dr Ashish U. Bisane
• Despite difficulty in identifying all the members of the oral microbiota and
understanding how they interact with each o...
Bacterial Culturing
• Gold Standard
• Plaque samples are cultivated under anaerobic conditions using
selective and non-sel...
Direct Microscopy
• Dark field or phase contrast microscopy.
• Most of the periodontal pathogens are non-
motile so it is ...
Immunodiagnostic Methods
• Immunologic assays employ antibodies that
recognize specific bacterial antigens to detect
targe...
Direct Immunofluoroscent
Assay (DFA)
AB conjugated
with Fluorescein
marker
Bacteria (Antigen)
Immunofluoroscent
complex de...
Indirect Immunofluoroscent
Assay
Primary
AB
Bacterial
AG
Immune
Complex
Secondary
Fluoroscein
conjugated
Antibody
Stains t...
Direct IFA
Indirect IFA
54Dr Ashish U. Bisane
Flow Cytometry or
Cytofluorography
Bacterial cells + species specific antibody
+ Secondary Fluorescein Conjugated
antibody...
ELISA
• Similar to Antibody and Antigen reaction, but the
fluorescence, instead of a radioisotope, is read using an
enzyma...
57Dr Ashish U. Bisane
58Dr Ashish U. Bisane
Latex Agglutination
• Based on binding of protein to latex.
Latex beads coated with species specific antibody
When beads c...
Enzymatic Methods
• Bacteria release specific enzymes.
• Certain group of species share common
enzymatic profile.
• E.g. T...
BANA Test
BANA Substrate
Hydrolysis by
Trypsin like
enzyme of bacteria
Hydrolysis
Formation of
Chromophore: B-
naphthylami...
Perioscan is a popular diagnostic kit uses
BANA reaction.
Disadvantages:
May be positive in clinically healthy site
Cann...
Molecular Biology for
Diagnosis
• Based on analysis of
DNA, RNA and
structure & function
of Protein of the
micro-organisms...
Nucleic Acid Probes
• A DNA probe uses a segment of a single
stranded DNA, labeled with a enzyme of a
radio isotope, that ...
Whole Genomic Probes
• Targets the whole DNA strand rather then a
specific sequence or gene.
• High chances to cross react...
66Dr Ashish U. Bisane
Oligonucleotide Probes
• Target variable region of 16sRNA or a specific
sequence in the DNA strand.
• Higher sensitivity a...
Hybridization
• Pairing of complementary strands of DNA
(nucleic acid) to produce a double stranded
DNA (nucleic acid).
• ...
Checkerboard DNA-DNA
Hybridization Technology
• Developed by Socransky et.al. 1994.
• 40 bacterial species can be detected...
• Advantages of DNA probes as compared to
bacterial culturing.
1. More sensitive and specific
2. Requires as less as 104 c...
Polymerase Chain Reaction
• Developed in 1985.
• Used almost universally to study DNA or RNA
obtained from a variety of ti...
72Dr Ashish U. Bisane
 Isolation of DNA from a fresh tissue specimen.
 Heating of complementary double strands of DNA.
 DNA splits into singl...
• In 1988, a thermo-stable DNA polymerase
isolated from the organism Thermus aquaticus
, known as Taq-polymerase was devel...
75Dr Ashish U. Bisane
Real Time PCRs
• To quantitatively assess target bacteria using
PCR technique.
• By using a single copy of the bacterial g...
• Advantages:
1. High degree of sensitivity and specificity
2. Reproducible and consistent
3. High detection limit. As les...
Advances in Characterizing
Host Response
• Asses host response by studying mediators as a
response to specific bacteria or...
GCF
• Most well studied, with almost 40 components in form
of host-derived enzymes, tissue breakdown products,
and inflamm...
Perio Paper Strips
Periotron 8000
80Dr Ashish U. Bisane
Saliva
• The next most used after GCF.
• Easily collected
• Contains both local and systemic derived markers for periodont...
Cytokines
• Are substances released by cells of the
immune system.
• Cytokines in GCF are: TNF-alpha, IL-1, IL-6,
and IL-8...
Host derived enzymes
1. Aspartate aminotrasnferase( AST),
2. Alkaline phosphatase,
3. B-glucoronidase,
4. Elastase,
5. Cat...
AST
• Derived from dead cells,.
• Elevated in periodontal disease
• Periogard is a commercially available colorimetric tes...
• ALP: released from osteoblast, neutrophils,
fibroblast..
• Beta Glucoronidase and Elastase: found in
Neutrophils.
• Beta...
Matrix metalloproteinases
• zinc and calcium dependent enzymes
• constitutively formed in the body, secreted by fibroblast...
Tissue Breakdown Products
• Analysis of GCF obtained from sites with
periodontitis clearly show elevated levels of
hydroxy...
Conclusion
• No proven marker available to predict the
disease activity as there is no proven
correlation between these ma...
Thank You !!
89Dr Ashish U. Bisane
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Advanced diagnostic aids 24 9-12

  1. 1. GOOD MORNING 1Dr Ashish U. Bisane
  2. 2. Advanced Diagnostic Aids 2By: Dr Ashish U. Bisane
  3. 3. Introduction • Periodontal disease is considered a site specific disease characterized by local inflammatory reaction to bacterial infection. • The attempts to identify specific bacteria, specific inflammatory cells or molecules, or specific breakdown products have been the soul reason for the advances in diagnostic methodology. 3Dr Ashish U. Bisane
  4. 4. Gingival Bleeding • Gingival Inflammation is diagnosed based upon: 1. Gingival Bleeding on Probing (Objective sign) 2. Gingival Redness 3. Gingival edema/swelling • Gingival bleeding is a good indicator of the presence of an inflammatory lesion in the connective tissue at the base of the sulcus and that the severity of bleeding increases with an increase in size of the inflammatory infiltrate. (Greenstein G, 1981; Polson AM, 1985) 5Dr Ashish U. Bisane
  5. 5. • To test for bleeding after probing, the probe is carefully introduced to the bottom of the pocket and gently moved laterally along the pocket wall. • Photomicrograph of thin, microulcerated epithelial lining of the soft tissue wall of an infected pocket. Such sites readily bleed when gently probed 6Dr Ashish U. Bisane
  6. 6. • Subjects who refrain from normal oral hygiene procedures have a resultant increase in plaque accumulation and demonstrate a concomitant increase in gingival bleeding as gingivitis develops over a 2-3 week period. (Greenstein G, 1981) • Lang et al 1991, in a retrospective study reported that sites that bled on probing at several visits had a greater probability of losing attachment than those that bled at one visit or did not bleed. • However, well controlled longitudinal studies investigated the predictive values of such clinical sign, trying to correlate them with attachment loss, but failed to demonstrate a significant correlation between bleeding on probing and other clinical signs and subsequent loss of attachment. (Badersten.A, 1985; Haffajee.A.D, 1983) 7Dr Ashish U. Bisane
  7. 7. • Drawbacks: 1. Healthy sites may bleed on probing as any force greater than 0.25N evokes bleeding even in an intact periodontium. 2. Relationship to disease progress is unclear. 3. Signs of inflammation are inhibited in smokers (Nair.P, 2003). 8Dr Ashish U. Bisane
  8. 8. (Sub)Gingival Temperature • Kung et al, 1990; demonstrated that the subgingival temperature at diseased sites is increased compared with healthy sites and that a natural anteroposterior temperature gradient exists within the dental arches. • Kung et al, 1990; thus, claimed that thermal probes are sensitive diagnostic devices for measuring early inflammatory changes in the gingival tissues. 9Dr Ashish U. Bisane
  9. 9. Perio-Temp (Thermal Periodontal Probing System) • Perio-Temp Probe (Abiodent) • Enables the calculation of temperature differential with a sensitivity of 0.10celcius. • Individual temperature differences are compared with those expected for each tooth, and higher temperature pockets are signaled with a red-emitting diode. 10Dr Ashish U. Bisane
  10. 10. Perio-Temp 11Dr Ashish U. Bisane
  11. 11. • 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. • Haffajee et al, 1992; also found that elevated subgingival site temperature was particularly related to attachment loss in shallow pockets and that P. intermedia, P. micros, P. gingivalis, T. forsythia and Aac had elevated proportions in the total microbiota in sites with elevated temperatures. • Trikilis.N, 1999; reported differences in the temperature differentials between subgingival temperature and sublingual temperature in smokers compared to non- smokers. 12Dr Ashish U. Bisane
  12. 12. • The reason why temperature increases with probing depth is unclear. A possible explanation is an increase in cellular and molecular activity caused by increased periodontal inflammation with increased probing depth. • Whether the pathogens are responsible for higher temperature by initiating the inflammatory process, or whether the increased temperature provides an environment favourable for the pathogens, remains unclear. 13Dr Ashish U. Bisane
  13. 13. Periodontal Probing • Currently the “Gold Standard” for recording changes in periodontal status in longitudinal measurement of clinical attachment levels. • Factors affecting probing measurements: (Listgarten. M. A, 1976) 1. Probing technique. 2. Probing force. 3. Size of the probe tip. 4. Angle of insertion of the probe. 5. Precision of probe calibration. 14Dr Ashish U. Bisane
  14. 14. Drawbacks in manual probing: 1. Use of the periodontal probe - many problems in terms of sensitivity and reproducibility of the measurements. 2. Clinical pocket depth obtained does not normally coincide with the histologic pocket depth. 3. Inflammed tissue offers less resistance to probe penetration, and the probe tip either coincides with or is apical to the coronal level of connective tissue attachment. (Listgarten. M. A, 1976) 4. Healed gingiva and post subgingival instrumentation, there is increased resistance to periodontal probing. (Magnusson. I, 1980) 15Dr Ashish U. Bisane
  15. 15. Probing Force • Penetration of probe has been positively correlated with probing force. (Mombelli.A, 1986) • With forces up to 30g, the tip of probe seems to remain within the intact junctional epithelium (Armitage. G.C, 1994) • Forces up to 50g are necessary to diagnose periodontal osseous defects (Kalkwarf.K.L, 1986) 16Dr Ashish U. Bisane
  16. 16. Classification • First generation probes/ conventional • Second generation probes/pressure sensitive • Third generation probes/ computerized Standardization of probe tip (<1mm) and use of registration stents have been suggested to maintain reproducible angulations and reduce the error. (Badersten.A. 1984) 17Dr Ashish U. Bisane
  17. 17. 18Dr Ashish U. Bisane
  18. 18. National Institute of Dental and Craniofacial Research (NIDCR) criteria for overcoming limitations of conventional periodontal probing (Parakkal. P.F, 1979) Limitation Conventional Probing NIDCR Criteria 1. Precision 1mm 0.1 mm 2. Range 12 mm 10 mm 3. Probing Force Non standardized Constant & Standardized 4. Applicability Non-invasive and easy to use Non-invasive , light weight and easy to use 5. Reach Easy to access any location around all teeth Easy to access any location around all teeth 6. Angulation Subjective A guidance system to ensure proper angulation. 7. Security Easily sterilized. Simple stainless steel instruments.. Complete sterilization of all the portions entering the mouth. No biohazard from material or electric shock. 8. Readout Depending upon voice dictation and recording in writing Direct electronic reading and digital output. 19Dr Ashish U. Bisane
  19. 19. Florida Probe System • Gibbs et al - developed Florida Probe System (Florida Probe Corporation) • Parts of Florida Probe System: 1. Probe handpiece 2. Digital readout 3. Foot switch 4. Computer interface 5. Computer • Probe tip - 0.4 mm in diameter. This probe tip reciprocates through a sleeve, and the edge of the sleeve provides a reference by which measurements are made. • These measurements are made electronically and transferred automatically to the computer when the foot switch is pressed. • Constant probing force is provided by coil spring inside the probe hand – piece and digital readout. 20Dr Ashish U. Bisane
  20. 20. Advantages 1. Constant probing force 2. Precise electronic measurement 3. Computer storage of data Disadvantages 1. Lack of tactile sensitivity - independent movement, which forces the operator to predetermine an insertion point and angle. 2. Use of a fixed force setting throughout the mouth, regardless of the site or inflammatory status, may generate inaccurate measurements or patient discomfort. 3. One common problem reported in different studies where the Florida Probe System has been compared with conventional probing is the underestimation of deep probing depths by the automated probe. (Perry. D.A. 1994) 21Dr Ashish U. Bisane
  21. 21. 22Dr Ashish U. Bisane
  22. 22. 23Dr Ashish U. Bisane
  23. 23. Florida Probe System to Record CAL • CALs are recorded relative to a fixed reference point, such as the occlusal surfaces of the teeth (Disk Probe) or a Prefabricated stent (Stent Probe). • These measurements are made sequentially over time, and thus differences in relative attachment levels at consecutive examinations must be calculated. 24Dr Ashish U. Bisane
  24. 24. 25Dr Ashish U. Bisane
  25. 25. Florida PASHA Probe • Modification of Florida probe to detect CEJ. • Has a Modified Sleeve, which includes a prominent 0.125 mm edge to facilitate a “catch” of the CEJ. • The width of this edge is considered small enough not to interfere with probing depth measurement, offering clinicians to measure CAL and probing depth concurrently. • Reliably reproduces and identifies CEJ in human skulls (Preshaw.P.M, 1999) and shows promise in measuring CALs in humans (Karpinia. K, 2004) 26Dr Ashish U. Bisane
  26. 26. Toronto Automated Probe • The uses the occlusal-incisal surface to measure relative clinical attachment levels. (Mcculloch.C.A.1991) • The sulcus is probed with a 0.5mm nickel- titanium wire that is extended under air pressure. • It controls angular discrepancies by means of a mercury-tilt sensor that limits angulation within + 30 degrees, but it requires reproducible positioning of patient’s head and cannot easily measure 2nd and 3rd molars. 27Dr Ashish U. Bisane
  27. 27. Interprobe & Perioprobe • An electronic probe using an optical encoder transduction element - evaluated by Goodson and Kondon. • These authors reported somewhat higher reproducibility with the electronic probe compared to conventional probing and a correlation coefficient of 0.82 between the two different methods. • This probe has now been named Interprobe (Bausch and Lomb). • They provide constant probing force, computer storage of data, and precise electronic management of the resulting inflammation. • Clinical evaluations of these systems- reported slightly improved probing, although not clinically significant 28Dr Ashish U. Bisane
  28. 28. Probes and the examination of patients considered for implants • Examination of patients - considered for implants includes both clinical evaluation of soft tissues and a radiographic evaluation.. • Probing around implants is difficult (1) the prosthetic construction may need to be removed for access (2) standard metal instruments are unsuitable. • Instead, plastic or titanium probe tips should be used to avoid damage of the implant / tissue interface. If automatic probing is considered, the Florida Probe is available with a titanium tip that will not hurt the implant ; also, the Interprobe system comes with disposable plastic tips. 29Dr Ashish U. Bisane
  29. 29. 30Dr Ashish U. Bisane
  30. 30. 31Dr Ashish U. Bisane
  31. 31. Drawbacks of Conventional Radiography • Conventional radiographs are very specific but lack sensitivity. • Considerable amount of bone loss should occur for being visible on the radiographs. • Causes of variability in conventional radiographic techniques: 1. Variations in projection geometry. 2. Variations in contrast and density caused by differences in film processing, voltage and exposure time. 3. Masking of osseous changes by other anatomic structures. 32Dr Ashish U. Bisane
  32. 32. Solutions for Standardization of Radiographic Techniques • Radiographs should be obtained in a constant reproducible plane using film holders with a template containing some type of template material, which is placed in a constant position on a group of teeth and an extension arm that can be precisely attached to both the film holder and the x-ray tube (Rosling. B. 1975) 33Dr Ashish U. Bisane
  33. 33. Digital Radiography • Digital radiography enables the use of computerized images - stored, manipulated, and corrected for under – and overexposures. • Yield almost equal image properties compared with conventional radiographs, but through digital storage and processing, diagnostic information can be enhanced. • Dose reduction obtained with this technique (between 1/3 to ½ of dose reduction compared with conventional radiographs). • Telediagnosis, video conferencing, rapid image transmission are some of the other advantages. 34Dr Ashish U. Bisane
  34. 34. Types of Digital Intraoral Radiographic Systems (Parks.E.T. 2002) • First system 1. Charge – Coupled Devices (CCDs) 2. Complementary Metal Oxide Semiconductor (CMOS) receptors as detectors. • These devices are placed in the patient’s mouth and are linked by a wire to the computer. • On radiation exposure the radiographic image appears on computer screen. • The detector is then moved to the next position and so on until the complete area to be imaged is covered. 35Dr Ashish U. Bisane
  35. 35. • Second System: uses Photostimulable Phosphor (PSP) plates as detectors. • 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. • PSP plates are placed and exposed similar to regular film. • The exposed plates are placed on a plate scanner and scanned by a laser beam, and the radiographic image appears on the computer screen. 36Dr Ashish U. Bisane
  36. 36. The Digital Advantage 1. Speed of image capture and display 2. Low x-ray exposure 3. Ability to manipulate the image and maximize diagnostic efficacy. 4. Use of digital tools such as linear, angular and density measurements. 5. Improved patient education 6. Ease of storage, transfer and copying. 7. Seamless integration with electronic patient record management or other software 38Dr Ashish U. Bisane
  37. 37. • Once captured and displayed, computer software can be used to enhance the digital image and increase its diagnostic efficacy. (Preston J D, 1999) • Exposure can be adjusted. • A sharpening (Edge Enhancement) filter increases the definition and separation of adjacent structures. • Inversion filters provide a negative of the image that might sometime reveal diseases not seen on the positive images. • Areas of interest can be magnified. • Different image qualities allow better detection of dental diseases. (High contrast for caries and Low contrast for Alveolar Crest detection) 39Dr Ashish U. Bisane
  38. 38. Subtraction Radiography • This technique requires a paralleling technique to obtain a standardized geometry and accurate superimposable radiographs. • It relies on the conversion of serial radiographs into digital images. • The serially obtained digital images can be superimposed and the resultant composite can be viewed on a video screen. • Changes in the density and volume of bone can be detected as 1. Lighter areas (Bone Gain) 2. Dark Areas (Bone Loss) • It facilitates both quantitative and qualitative visualization of even minor density changes in bone by removing the unchanged anatomic structures from the image. 40Dr Ashish U. Bisane
  39. 39. Advantages: High correlation between alveolar bone loss and CAL changes Increased detection of small osseous lesion Both quantitative and qualitative visualization More sensitive 41Dr Ashish U. Bisane
  40. 40. • Studies using this technique have shown: 1. A higher degree of correlation between changes in alveolar bone determined by subtraction radiography & CAL changes in periodontal patients after therapy. (Hausmann E, 1985) 2. Increased detectability of small osseous lesions compared with conventional radiographs from which the subtraction images are produced. (Rethman M, 1985) 42Dr Ashish U. Bisane
  41. 41. Serial radiographs converted to digital images superimposed composite image Quantitative changes 43Dr Ashish U. Bisane
  42. 42. • The disadvantage is the need to be almost at identical projection alignment during the exposure of sequential radiographs which makes this method impractical in clinical setting. 44Dr Ashish U. Bisane
  43. 43. Diagnostic/Digital Subtraction Radiography (DSR) • Combines the use of a positioning device during film exposure with a specialized software designed for digital image subtraction using conventional personal computers in dental offices. • The software corrects the effects of angular alignment discrepancies and provides some degree of flexibility in the imaging procedure. • DSR showed statistically significant gains in diagnostic accuracy over conventional radiographs but no differences with Subtraction radiography. (Nummikoski PV, 2000) 45Dr Ashish U. Bisane
  44. 44. Computer Assisted Densitometric Image Analysis System (CADIA) • A video camera measures the light transmitted through the radiograph and signals from the camera are converted into gray scale images (Bragger U, 1988) • The camera is interfaced with an image processor and a computer that allows the storage and mathematic manipulation of the images. • CADIA offers an objective method for following alveolar bone density changes quantitatively over time. • It has also shown higher sensitivity and a higher degree of reproducibility and accuracy than DSR. 46Dr Ashish U. Bisane
  45. 45. Advances in Microbiological Analysis 47Dr Ashish U. Bisane
  46. 46. • Despite difficulty in identifying all the members of the oral microbiota and understanding how they interact with each other and with the host, a limited number of micro-organisms have demonstrated a clear etiologic role and have been identified as periodontal pathogens. (Genco RJ, 1996) • Microbiologic tests to identify the putative organisms have the potential to support the diagnosis of various forms of periodontal disease, to serve as indicators of disease initiation and progression (i.e. disease activity) and to determine which periodontal sites are at higher risk for active destruction. • Microbiologic tests can also be used to monitor periodontal therapy directed at the suppression or eradication of periodontal pathogenic micro-organisms.(Van Winkelhoff AJ, 1996) 48Dr Ashish U. Bisane
  47. 47. Bacterial Culturing • Gold Standard • Plaque samples are cultivated under anaerobic conditions using selective and non-selective media. • Advantage: 1. Relative and Absolute count of the cultured species. • Disadvantage: 1. Strict sampling conditions 2. Difficulty in culturing most organisms 3. Low sensitivity : organisms lesser then 103 is difficult to detect 4. Time consuming 5. Expensive equipment and experienced personnel 49Dr Ashish U. Bisane
  48. 48. Direct Microscopy • Dark field or phase contrast microscopy. • Most of the periodontal pathogens are non- motile so it is difficult to identify. • Even different species of Treponema cannot be distinguished by direct microscopic techniques. 50Dr Ashish U. Bisane
  49. 49. Immunodiagnostic Methods • Immunologic assays employ antibodies that recognize specific bacterial antigens to detect target micro-organisms. 1. Direct and Indirect Immunofluoroscent (microscopic) Assays 2. Flow Cytometry 3. Enzyme Linked Immunosorbent Assay (ELISA) 4. Latex Agglutination 51Dr Ashish U. Bisane
  50. 50. Direct Immunofluoroscent Assay (DFA) AB conjugated with Fluorescein marker Bacteria (Antigen) Immunofluoroscent complex detectable under microscope 52Dr Ashish U. Bisane
  51. 51. Indirect Immunofluoroscent Assay Primary AB Bacterial AG Immune Complex Secondary Fluoroscein conjugated Antibody Stains the primary AG:AB complex 53Dr Ashish U. Bisane
  52. 52. Direct IFA Indirect IFA 54Dr Ashish U. Bisane
  53. 53. Flow Cytometry or Cytofluorography Bacterial cells + species specific antibody + Secondary Fluorescein Conjugated antibody Introduced in flowcytometer. Bacterial cells are separated into single cell suspension by means of laminar flow. Cells identified by lasers. 55Dr Ashish U. Bisane
  54. 54. ELISA • Similar to Antibody and Antigen reaction, but the fluorescence, instead of a radioisotope, is read using an enzymatically driven color reaction with a photometer. • Intensity of color depends upon the concentration of the antigen and is usually read photometrically for optimal quantification.  Evalusite: commercially available chairside diagnostic kit to detect Aa, Pg and Pi.  It involves linkage between antigen and a membrane bound antibody to form an immunocomplex that is revealed later with a colorimetric reaction. 56Dr Ashish U. Bisane
  55. 55. 57Dr Ashish U. Bisane
  56. 56. 58Dr Ashish U. Bisane
  57. 57. Latex Agglutination • Based on binding of protein to latex. Latex beads coated with species specific antibody When beads come in contact with specific species in sample they bind and agglutination occurs Clumping of beads is visible Test positive 59Dr Ashish U. Bisane
  58. 58. Enzymatic Methods • Bacteria release specific enzymes. • Certain group of species share common enzymatic profile. • E.g. Tf, PG, Td, and Capnocytophaga species release trypsin-like enzyme. • The activity of this enzyme is measured with the hydrolysis of the colorless substrate:  N-benzoyl-d L-arginine-2-naphthylamide (BANA) 60Dr Ashish U. Bisane
  59. 59. BANA Test BANA Substrate Hydrolysis by Trypsin like enzyme of bacteria Hydrolysis Formation of Chromophore: B- naphthylamide Addition of Fast Garnet The chromophore turns orange red 61Dr Ashish U. Bisane
  60. 60. Perioscan is a popular diagnostic kit uses BANA reaction. Disadvantages: May be positive in clinically healthy site Cannot detect disease activity Limited organisms detected Other pathogens may be present if it’s negative. 62Dr Ashish U. Bisane
  61. 61. Molecular Biology for Diagnosis • Based on analysis of DNA, RNA and structure & function of Protein of the micro-organisms. • A “probe” is a known nucleic acid molecule (DNA or RNA) from a specific micro- organism artificially synthesized and labeled for its detection when placed with a plaque sample. 63Dr Ashish U. Bisane
  62. 62. Nucleic Acid Probes • A DNA probe uses a segment of a single stranded DNA, labeled with a enzyme of a radio isotope, that is able to hybridize to a complimentary nuclei strand, and thus detect presence of target microorganism. • Two types: 1. Whole genomic probes 2. Oligonucleotide probes 64Dr Ashish U. Bisane
  63. 63. Whole Genomic Probes • Targets the whole DNA strand rather then a specific sequence or gene. • High chances to cross react with non target microorganism • Lower sensitivity and specificity. • Whole genomic probes for Aa, Pg, Pi and Td have been commercially developed and tested E.g.DMDx, Omnigene. 65Dr Ashish U. Bisane
  64. 64. 66Dr Ashish U. Bisane
  65. 65. Oligonucleotide Probes • Target variable region of 16sRNA or a specific sequence in the DNA strand. • Higher sensitivity and specificity. • These bacterial 16s RNA genes contain both regions shared by different bacteria and short stretches of variable regions shared by only specific micro-organisms of the same species or genus. (Moncla BJ, 1990) 67Dr Ashish U. Bisane
  66. 66. Hybridization • Pairing of complementary strands of DNA (nucleic acid) to produce a double stranded DNA (nucleic acid). • All hybridization methods use radiolabeled or fluoroscence-labeled DNA probes that bind to the target DNA of interest, thus allowing its visualization. 68Dr Ashish U. Bisane
  67. 67. Checkerboard DNA-DNA Hybridization Technology • Developed by Socransky et.al. 1994. • 40 bacterial species can be detected using whole genomic digoxigenin-labeled DNA probes. • Large number of samples can be tested and up to 40 oral species detected with a single test. • Highly specific test. • Requires expensive sophisticated laboratory equipment. 69Dr Ashish U. Bisane
  68. 68. • Advantages of DNA probes as compared to bacterial culturing. 1. More sensitive and specific 2. Requires as less as 104 cells of each species to be detected. 3. Multiple species detected with a single test 4. Does not require viable bacteria 5. Large number of samples can be assessed. • Disadvantages: 1. Expensive 2. Expert personnel to carry out the test 3. Not easily available 70Dr Ashish U. Bisane
  69. 69. Polymerase Chain Reaction • Developed in 1985. • Used almost universally to study DNA or RNA obtained from a variety of tissue sources. • Involves amplification of a region of DNA by a primer specific to the target species. • If there is amplification then it indicates the presence of the target species in the sample. 71Dr Ashish U. Bisane
  70. 70. 72Dr Ashish U. Bisane
  71. 71.  Isolation of DNA from a fresh tissue specimen.  Heating of complementary double strands of DNA.  DNA splits into single stranded forms.  Single stranded forms are intended to act as template dictating the nucleotide sequencing in vitro.  Amplification is done using a DNA polymerase that requires a primer or a known short oligonucleotide sequence complementary to the border of the region that is to be amplified.  To obtain amplified fragments of constant length and in large quantities, a second primer, complementary of the opposed chain must be used to bind the template and flank the region of interest.  This amplification can be performed several number of times known as cycles.  This sequenced DNA is then detected and visualized through electrophoresis in aragose gel and ethidium-bromure obtaining a qualitative signal. 73Dr Ashish U. Bisane
  72. 72. • In 1988, a thermo-stable DNA polymerase isolated from the organism Thermus aquaticus , known as Taq-polymerase was developed. • This Taq-polymerase has allowed automatization of the reaction using specific appliances called thermocycles. 74Dr Ashish U. Bisane
  73. 73. 75Dr Ashish U. Bisane
  74. 74. Real Time PCRs • To quantitatively assess target bacteria using PCR technique. • By using a single copy of the bacterial genes per cell, a good correlation between the fluorescent signal measured and the number of cells can be obtained. (Lyons SR,2000; Smith AJ, 1996) 76Dr Ashish U. Bisane
  75. 75. • Advantages: 1. High degree of sensitivity and specificity 2. Reproducible and consistent 3. High detection limit. As less as 5- 10 cells can be amplified and detected. 4. Less cross reactivity under optimal conditions. 5. Many species can be detected simultaneously. • Disadvantage: 1. Expensive lab equipments 2. Small quantity needed for reaction may not contain the necessary target DNA. 3. Plaque may contain enzymes which may inhibit these reactions. 77Dr Ashish U. Bisane
  76. 76. Advances in Characterizing Host Response • Asses host response by studying mediators as a response to specific bacteria or local release of inflammatory mediators or enzymes as response to infection. • Source of samples may be; GCF, Saliva, or Blood. • GCF is most commonly used, where as saliva is been recently been researched recently. 1. Host derived enzymes 2. Inflammatory mediators and products 3. Tissue Breakdown Products 78Dr Ashish U. Bisane
  77. 77. GCF • Most well studied, with almost 40 components in form of host-derived enzymes, tissue breakdown products, and inflammatory mediators. • Collected with paper strips, micro papillary tubes, micropipettes, microsyringes, plastic strips. • Paper strips commonly used, introduced in sulcus for 30 secs and volume is measured using Periotron 6000. • Periotron measures the capacitance across the wet paper strip, which is converted to digital reading. • Periotron reading have high correlation with clinical gingival indices. • Quickest and easiest way to measure GCF. 79Dr Ashish U. Bisane
  78. 78. Perio Paper Strips Periotron 8000 80Dr Ashish U. Bisane
  79. 79. Saliva • The next most used after GCF. • Easily collected • Contains both local and systemic derived markers for periodontal diseases. • Collected from parotid, sub-mandibular or sub lingual or as ‘Whole saliva’. • Whole saliva contains secretions of major and minor salivary glands, desquamated cells, and GCF. • Markers to look for in saliva: 1. Proteins and enzymes from host, 2. Phenotypic markers, 3. Host cells, 4. Hormones, 5. Bacteria, bacterial products, 6. Volatile compounds, and ions. 81Dr Ashish U. Bisane
  80. 80. Cytokines • Are substances released by cells of the immune system. • Cytokines in GCF are: TNF-alpha, IL-1, IL-6, and IL-8 • Can be used to determine the disease activity. • Esp. Prostaglandin E in increased in GCF of periodontitis patients. • Can be used to determine disease activity 82Dr Ashish U. Bisane
  81. 81. Host derived enzymes 1. Aspartate aminotrasnferase( AST), 2. Alkaline phosphatase, 3. B-glucoronidase, 4. Elastase, 5. Cathespins, 6. MMPs. 83Dr Ashish U. Bisane
  82. 82. AST • Derived from dead cells,. • Elevated in periodontal disease • Periogard is a commercially available colorimetric test for AST. • It involves GCF collection with a filter paper strip, which is then placed in a tromethamine hydrochloride buffer. A substrate reaction mixture containing L-aspartic and a- ketoglutaric acids are added and allowed to react for 10min. In presence of AST, aspartate and glutarate are catalyzed to oxaloacetate and glutamate. Addition of a dye (Fast Red) results in a colour product. The intensity of which is proportional to the AST activity in GCF sample. • However, AST levels cannot differentiate active and inactive sites. 84Dr Ashish U. Bisane
  83. 83. • ALP: released from osteoblast, neutrophils, fibroblast.. • Beta Glucoronidase and Elastase: found in Neutrophils. • Beta Glucoronidase, Elastase, Neutral protease, and Cathepsins: All are shown to be higher in diseased sites. May be used to predict severity of disease or to predict disease activity. 85Dr Ashish U. Bisane
  84. 84. Matrix metalloproteinases • zinc and calcium dependent enzymes • constitutively formed in the body, secreted by fibroblast and macrophages. • Normally help in degrading and remodeling of extracellular matrices. • In chronic periodontitis they cause the degradation of the collagen fibrils in PDL and Alveolar bone. • MMP,2,3,8 9 and 13 play important role. • MMP8 level is associated with the attachment loss • Level reduces in response to treatment. (Chair side test kit) • Can be used to indicate present disease status and predictor of future disease. 86Dr Ashish U. Bisane
  85. 85. Tissue Breakdown Products • Analysis of GCF obtained from sites with periodontitis clearly show elevated levels of hydroxyproline from collagen breakdown and GAGs from matrix degradation. (Last KS, 1985) • Osteocalcin and Type-I collagen peptides. 87Dr Ashish U. Bisane
  86. 86. Conclusion • No proven marker available to predict the disease activity as there is no proven correlation between these markers and the clinical loss of attachment. • Search of tool with high predictive value, simple, safe and cost effective still needed. 88Dr Ashish U. Bisane
  87. 87. Thank You !! 89Dr Ashish U. Bisane
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