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Advanced diagnostic aids

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Brief discussion about diagnostic aids in periodontics

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Advanced diagnostic aids

  1. 1. ADVANCED DIAGNOSTIC AIDS IN PERIODONTICS SHEETHALAN M S R
  2. 2. DIAGNOSIS • WHO definition –it is the art of chronological organization and critical evaluation of the information obtained of patients history , lab investigations, clinical examination so as to identify the disease type and etiology. • Greek word – • Dia =through • Gnosis =to know
  3. 3. WHY IT IS IMPORTANT ??? • Diagnostic testing should be considered as an aid to the diagnostic process. • Proper diagnosis is required for the rational treatment and preventive strategy
  4. 4. PRINCIPLES OF DIAGNOSTIC TESTING • Sensitivity • Specificity • Predictive value • Positive • Negative
  5. 5. PERIODONTAL DIAGNOSTIC TESTS • Clinical methods • Radiographic methods • Microbial analysis • Host response
  6. 6. CLINICAL METHODS • Gingival bleeding • Periodontal probing • Tooth mobility • Gingival temperature • Halitosis
  7. 7. BLEEDING ON PROBING • Bleeding on probing is widely regarded as a relatively objective sign of gingival inflammation since it is either present or absent • According to Lang et al any force greater than 0.25N may evoke bleeding at healthy sites with an intact periodontium. • possible predictor of the progression of periodontitis.
  8. 8. • Several studies have shown that gingival bleeding is a sensitive clinical indicator of early gingival inflammation (Mombelli and Graf). • It has also been shown that gingival bleeding is a good indicator of the presence of an inflammatory lesion in the connective tissue at the base of the sulcus and that severity of bleeding increases with an increase in size of the inflammatory infiltrate (Greenstein, Caton, Polson).
  9. 9. • Lang et al in a retrospective study reported that sites that bled on BOP at several visits had a higher probability of losing attachment than those that bled on 1 visit or did not bleed at all. • However, other well-controlled longitudinal studies failed to demonstrate a significant correlation between BOP and other clinical signs and subsequent attachment loss (Haffajee et al). • BOP is a good risk indicator of disease activity but not a good predictor.
  10. 10. GINGIVAL TEMPERATURE • Increased heat is generated by inflamed tissue because of increased blood flow and high metabolic rates associated with inflammatory processes. • Haffajee et al in 1992 reported on a site basis, increased mean subgingival temperatures have also been associated with deeper probing depths and greater levels of clinical attachment loss. 2 different rationales support these relationships: • Endotoxins of the infecting bacteria, especially the LPs of gram –ve organisms are exogenous pyrogens that stimulate macrophages to release endogenous pyrogens producing fever (Bencsics et al,1995) • Bacteria respond to changes in environmental temperature with changes in their growth rate, metabolic activities and expression of virulence factors (Maurelli et al, 1989)
  11. 11. • Studies with this probe have indicated that pockets with higher subgingival temperature usually bleed on probing and harbor elevated % of periodontal pathogens. Haffajee et al used this probe to asses its predictability in identifying loss of attachment, concluding that sites with a red temperature indication had more than twice the risk for future attachment loss than did those with a green indication.
  12. 12. PERIOTEMP PROBE (ABIODENT, INC., DANVERS, MASS): • It is a commercially available device that resembles a periodontal probe & used to measure subgingival temp to a precision of 0.1º C. • The temperature probe is inserted into a pocket to determine if a site is warmer than normal. A coloured reading indicates the temperature – green for cool, red for hot and amber when the recorded temperature is between cool and hot.
  13. 13. PERIODONTAL PROBING • Periodontal probe was the first attempt to quantify the data documenting the severity of periodontal disease. • G.V. Black was the first to describe the use of probe to explore the periodontal pockets. • It is used for examining the gingival sulcus, to assess the periodontal destruction, measure PD and CAL, elicit gingival bleeding, consistency of gingiva, furcation defects and to determine presence of subgingival calculus.
  14. 14. FACTORS AFFECTING THE ACCURACY OF PERIODONTAL PROBING (LISTGARTEN) • Size of the probe or the probe tip thickness. • Precision of probe calibration. • Inflammatory state of the tissues. • Contour of the tooth and root surface (particularly in furcations). • Angle of insertion of the probe. • Probing force. • Probing technique • Inter and intra examiner reliability. • All these variables contribute to a large standard deviation of 0.5 to 1.3 mm in clinical probing (Haffajee & Socransky).
  15. 15. GENERATIONS OF PERIODONTAL PROBING • 1st generation : the usual clinical instrument, a thin tapering tine marked probe to be read in mm. • 2nd generation/constant force probes : As above, but with a spring or electronic cutout when appropriate force is used. • 3rd generation/automated probes : Here when the probe is in place with specified force a device is activated that reads the measurement accurately. • 4th generation/3 dimensional probes: Currently under development are aimed at recording sequential probe positions along the gingival sulcus. • 5th generation/non-invasive 3 dimensional probes: These probes add ultrasound or other device to a 4th generations probes.
  16. 16. 1ST GENERATION
  17. 17. 2ND GENERATION • To overcome the disparity encountered due to change in probing force, the 2nd generation probes were evolved. • Studied have shown that forces with upto 30 g, the tip of the probe seems to remain within the junctional epithelium and forces of upto 50g are necessary to diagnose periodontal osseous defects (Kalkwarf et al).
  18. 18. HISTORY • Gabathuler and Hassell (1971) in a study designed to quantitate gentle probing, developed the first pressure sensitive probe. • Armitage et al (1977) designed a pressure sensitive probe holder to standardize the insertion forces. • Velden and de Vries (1978) developed a pressure sensitive probe which worked on air pressure system. • Vitek et al (1979) designed a leaf spring force controlled periodontal probe. This instrument delivers a force within 0.5 gms to a Michigan ‘O’ periodontal probe tip with terminal diameter of 0.35±0.05 mm.
  19. 19. • Tromp et al (1979) designed a probe to increase the reproducibility of pocket depth measurements. A constant torque spring was attached to a loose probe head, which could rotate in a point bearing. • van der Velden and de Vries (1980) modified the pressure sensitive probe in order to eliminate incorrect reading of the scale of the probe. The pressure was provided with a displacement transducer the electronic pocket depth readout.
  20. 20. • True Pressure Sensitve (TPS) probe (Vivacare): This true pressure sensitive plastic periodontal probe was introduced by Hunter F (1994). It has a disposable probing head. Probe tip is designed as a hemisphere with a diameter of 0.5mm and a rim surrounding the side of a ball, which aid in detection of CEJ, calculus, irregularities of root form and overhangs. • Controlled probing force to the probe tip was provided using a parallelogram. The probe also has a visual guide, a sliding scale where 2 marks meet.
  21. 21. 3RD GENERATION • These probes combine controlled force application, automated measurement and computerized data capture. • These were developed to reduce the measurement variation by standardizing the probing force, simplify reading the probe, helps in data recording and calculate the attachment level.
  22. 22. FLORIDA PROBE (FLORIDA PROBE CORP. GAINESVILLE, FLORIDA) • By university of fluoride. • Loss of attachment level can be detected to a certain by 99% with less than 1mm change. • The system consists of • A probe hand piece –common Michigan probe with Williams markings • A digital readout • Foot switch • Computer interface and a computer
  23. 23. FOSTER MILLER PROBE (FOSTER MILLER CORP. BOSTON, USA) • Capable of detecting the catch at the cemento enamel junction. • As the probe moves along the root surface , experience a rapid change in acceleration when the CEJ is crossed and while reaching the base of the pocket. • The working end of the probe is similar to the Michigan O periodontal probe in size and shape. • The extreme end of the tip was enlarged to approximately 0.5 mm in diameter in order to facilitate detection of the CEJ.
  24. 24. TORONTO AUTOMATED PROBE(HEIDENHAIN CORP., ILLINOIS) • The automated probe consist of a digital length gauge connective to nickel titanium alloy of 0.5mm diameter enclosed in polyethylene sheath. • Wire serves as a probe which is propelled by air pressure into the gingival sulcus with a regulated force. • The data is recorded by a microcomputer interfaced to the digital length gauge. • Measures attachment level in 0.1 mm gradations.
  25. 25. VARIOUS OTHER AUTOMATED PROBES • Peri Probe Comp (PD International AB, Sweden) • Acubek (Fiber Optic) Probe or Goodson probe • Interprobe (Bausch and Lomb, Tucker, GA) • Hunter / vivacare TPS Probe • Borodontic Probe
  26. 26. • All controlled forces probes are not the same ,with varying designs and operational differences. • Some might give more reproducible results than others. • There is insufficient data to completelty resolve the question of which controlled for probe gives the most reproducible results.
  27. 27. 4TH GENERATION • Fourth-generation refers to three dimensional(3D) probes. Currently under development, these probes are aimed at recording sequential probe positions along the gingival sulcus. • They are an attempt to extend linear probing in a serial manner to take into account the continuous and 3D pocket being examined.
  28. 28. 5TH GENERATION • 3D and noninvasive • aim to identify the attachment level without penetrating it. • only fifth-generation probe available, the UltraSonographic (US)probe (Visual Programs, Inc, Glen Allen,VA)
  29. 29. PROBES FOR FURCATION INVOVLEMENT • Naubers probe
  30. 30. PROBES FOR CALCULUS DETECTION • Detec Tar probe • Detect calculus by means of audio readings and are reported to increase chances of subgingival calculus detection. • Can be auto claves ,produces an audible beep to signify calculus detection.
  31. 31. EVIDENCES
  32. 32. TOOTH MOBILITY • Tooth mobility has been considered and investigated as an indirect measure of the functional condition of the periodontium as well as possible aggravating co-factor for periodontal disease. • This is evidenced by the large number of devices and method of tooth mobility assessment that have been developed and tested.
  33. 33. ASSESSMENT 1. Elbrecht’s indicator (1939) 2. Werner’s Oscillator (1942) 3. Dreyfus vibrator (1947) 4. Zinrner’s oscillograph (1949) 5. Manly’s device (1951) 6. Muhlemann’s Macro-periodontometer and Micro-periodontometer, Pictons gauge (1957) 7. Parfitt’s transformer (1958) 8. Joel’s technique (1958) 9. Goldber’s device (1961) 10. Korber’s transducers, USAFSAM periodontometer (O’Leary and Rudd 1963) 11. Pameijer’s device (1973) 12. Laser method (Ryden 1974) 13. Persson and Svensons device (1980) 14. Periotest (Schulte 1987, Simons AG, Germany)
  34. 34. TEST FOR TOOTH MOBILITY Periotest: • Schulte in collaboration with Siemens company developed an instrument designed to measure the mobility of the implants and natural teeth. This device rapidly percusses the tooth (16 times, 4 times a second) and then electronically records the rebound alteration pattern. The degree of attenuation (scale ranges from -8 to +50) is recorded digitally and acoustically then scaled into 4 degrees of tooth mobility.
  35. 35. • -8 to +9 : clinically firm tooth • 10-19 : palpable mobility • 20-29 : visible mobility • 30-50 : mobility in response to lip and tongue movements
  36. 36. • Goodson (1988) confirmed the correlation between PTV and clinical mobility index (MI). • The greater the alveolar bone height, the lower the periotest value.
  37. 37. HALITOSIS • Halitosis or oral malodor is most often caused by gram negative anaerobic bacteria which degrade the proteins and produce volatile sulfur compounds. These sulfur compounds can be detected by gas chromatography or a recently developed halimeter.
  38. 38. • ORAGANOLEPTIC RATING • GAS CHROMATOGRAPHY • DARK FIELD MICROSCOPY • SALIVA INCUBATION
  39. 39. • Halimeter (Interscan) developed by Rosenberg et al (1991) is used to diagnose halitosis. • It is a hydrogen sulfide portable analyzer which detects hydrogen sulfide, methyl mercaptan and less sensitive to dimethyl sulfide.
  40. 40. • Diamond Probe / Perio 2000 system (Diamond General Development Corporation, Ann Arbor, USA): A recently developed commercially available instrument designed so that it combines the features of a periodontal probe with the detection of volatile sulfur compounds in the periodontal pocket.
  41. 41. RADIOGRAPHS • Dental radiographs such as bite-wing, periapical and panoramic, are the traditional methods used to assess the destruction of alveolar bone associated with periodontitis. • Although radiographs do not accurately define the bone morphology buccally and lingually, they provide useful information on interproximal bone levels, root length, root proximity, periapical lesions and the remaining alveolar bone. • However, more than 30% of the bone mass at the alveolar crest must be lost for a change in bone height to be recognized on radiographs.
  42. 42. • Therefore, conventional radiographs are very specific but lack sensitivity. The low degree of sensitivity is mainly due to: • Variations in projection geometry • Variations in contrast and density due to differences in film processing, voltage and exposure time • Masking of osseous changes by other anatomic structures.
  43. 43. LIMITATION • Only 2 dimentional images. • Errors due to exposure and processing.
  44. 44. DIGITAL RADIOGRAPHY • The application of computer technology to radiography has allowed for image acquisition ,manipulation, storage ,retrieval and transmission. • Components • Detector • Digital converter • Computer for image display and storage
  45. 45. DETECTOR • Can be either charged coupled device(ccd) and photostimulable phosphoric plates. • A CCD consist of a chip of pure silicon with an active area called pixels. • Following exposure to radiation charges stored by the individual pixels are sequentially removed electronically, creating an output signal whose voltage is proportional to the charge on the pixels. • Output obtained by using anague to digital converter.
  46. 46. • Can be • Direct – images obtained from the CCD are immediately displayed in the output. • Indirect – uses radiographic film as the image receptor which is scanned to view the radiograph
  47. 47. ADVANTAGES OF RVG • Immediate image display • Ability to manipulate the image contrast. • Xray dosage reduction of 60% when compared with e-speed film and 77% compared to D-speed film. DISADVANTAGES • Limited sensor area • Decreased image resolution
  48. 48. SUBTRACTION RADIOGRAPHY: • Grondahl and Grondahl (1983) who introduced this technique in periodontal diagnosis. Later, Jeffcoat (1992) used this technique in the determination of periodontal disease progression. • This technique relies on the conversion of serial radiographs into digital images.
  49. 49. • In order to digitize a radiograph , the computer digitizer automatically superimposes a grid over the radiograph and converts the gray level of radiograph within each box of the grid to a number from 0 (black) to 225 (white). • Serially obtained digital images can then be super imposed and the resultant composite viewed on a videoscreen.
  50. 50. EVIDENCE • Jeffcoat et al(1990) showed a strong correlation between probing attachment loss using sequential measurements made with automated probe and bone loss detected with digital subtraction radiography. • Christgan et al (1998) evaluated the ability of quantitative digital subtraction radiography to detect small changes in bone thickness adjacent to the tooth roots. High correlation was found between objective ,quantitative assessment of subtle changes in the alveolar bone.
  51. 51. COMPUTER ASSISTED DENSITOMETRIC IMAGE ANALYSIS (CADIA): • By Bragger et al (1988) • The radiographs are viewed by a video camera linked to an image processor, digitized and the image displayed on the screen of the analyzer. • Tooth root and alveolar bone height can be measured to an accuracy of 0.01 mm. it is the most sensitive method of visualizing the alveolar crest- CEJ and measuring the radiographic bone loss in periodontal surgical site. • Deas et al demonstrated that the prevalence of progressing lesions in periodontitis, as detected by this radiographic method , may be much higher than previously thought.
  52. 52. • Brager et al (1989) tested the applicability of CADIA for quantitative assessment of alveolar bone density changes in furcation of multi rooted teeth.In 21 patients radiographs were taken after flap surgery.Results indicate that CADIA may give valuable additional diagnostic information regarding alveolar bone density. • Woo et al (2003) caliberated and validated a digital subtraction radiography system using scanned images obtained from CADIA,suggesting that the system could be suitable for detection of small changes in alveolar bone.
  53. 53. ADVANTAGES • Even minordensity in the bone can be assessed by this technique. • Most useful for determining the bone changes in periodontal examination or bone around dental implants are stable. DISADVANTAGES • Requires identical alignment of X-ray machine, teeth and film on each occasion.
  54. 54. COMPUTER TOMOGRAPHY: • Godfrey Hounsfield in 1972 invented a revolutionary imaging technique i.e. CT and claimed it to be 100 times more sensitive than the conventional x-rays. • ADVANTAGES- • CT completely eliminates the superimposition of images of structures superficial or deep to the area of interest within the patient. • Because of the inherent high contrast resolution of CT, differences may be distinguished between tissues that differ in physical density by less than 1% • Multiplanar images
  55. 55. • Consist of radiographic tube emitting a fine collimated fan shaped x-ray beam directed to a series of scintillation detectors. • Depending on the geometry of the scanner, both the radiographic tube and the detectorsmay rotate synchronously about the patient or detectors may form a continuous ring about the patient and the x-ray tube. • The CT image is reconstructed by the computer,which mathematically manipulate the transmission data obtained from multiple projections. • Recent advance is use of CBCT in dental imaging where x-rays are divergent form a cone.
  56. 56. MAGNETIC RESONANCE IMAGING (MRI) : • 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 profiles of the tissues. • Mainly used in the study of TMJ and the soft tissue lesion of gingiva and other oral structures.
  57. 57. • To acquire MRI images ,pt places in a strong magnetic field. The protons of the hydrogen nuclei of the water within the tissues rotate like a spinning top about the direction of the magnetic field. • Resonance frequency energy is applied and then removed. The response of the nuclei to the resonance frequency stimulation is observed in a receiver coil. • Mathematical algorithms then reconstruct slices or planar images of the MRI appearances of the organs of interest.
  58. 58. ULTRASONOGRAPHY : Ultrasonography, a useful noninvasive and painless procedure, has been used as a diagnostic tool in dentistry for the examination of dental hard tissues, PDL space, determination of alveolar bone morphology, detection of carious lesions in the enamel and the measurement of furcation involvement.
  59. 59. • Frequencies range from 1-20 MHz. • Scanners used for sonography generate electrical impulses that are converted into ultra high frequency sound waves by a transducer. • As the ultrasound waves passes through the tisues of interest it is attenuated by a combination of absortion, reflection ,refraction ,and diffusion. • Sonic waves that are reflected back towards the transducer causes a change in the thickness of the piezoelectric crystals, which inturn produce an electrical signal that is amplified ,processed and ultimately displayed in the monitor.
  60. 60. NUCLEAR MEDICINE : • In this technique, a short-lived radiolabeled bone seeking radio-pharmaceutical uptake (BSRU), such as diphosphonate compound, which is labeled radionuclide Technitium 99m. this technique is fairly accurate also in predicting subsequent bone changes and has the potential to provide an immediate measure of disease activity. • Progressive disease can be identified before bone loss is evident on conventional radiographs.
  61. 61. • Kaplan et al (1975) observed that beagle dogs with moderate to advanced alveolar bone loss had a six times grater alveolar bone seeking radio pharmaceutical uptake (BSRU) compared to dogs without alveolar bone loss, indicating its applicability in periodontics. • Jeffcoat et al in 1980s examined single measurement of BSRU around the teeth correlated well with the radiographic loss of bone. • Goldhaber and coworkers (mid 90s) began to apply nuclear medicine technique to study of periodontal bone resorption.
  62. 62. • Nuclear medicine technique has been used in limited human patients setting to determine the disease activity. • Due to additional risk of radiation in patients, nuclear medicine is not currently applicable to clinical practice.
  63. 63. MICROBIOLOGICAL ANALYSIS • Bacterial plaque plays a primary role in the initiation and progression of periodontal diseases. • A no. of assays have been developed for their detection and relative quantification in patient plaque samples. • It should be known that these assays themselves are not diagnostic for periodontal diseases. They indicate that the presence of these organisms can increase a subjects risk for periodontal attachment loss.
  64. 64. SAMPLES • Selecting the proper specimen site and collecting an adequate sample are essential features in periodontal microbiology. • Samples from the oral mucosa or from saliva are usually obtained with sterile paper points, or swabs and then transferred directly into an appropriate anaerobic transport medium. • In order to obtain a true sample of subgingival plaque, it is first necessary to remove all traces of supragingival plaque. The subgingival plaque can then be removed either with a sterile curette or with a sterile paper point. This is then rapidly transferred to the anaerobic transport medium.
  65. 65. • Culture techniques • Dark ground or phase contrast microscopy • Immunological assays • DNA Probes • Enzyme based assays
  66. 66. CULTURE TECHNIQUES • Culture techniques are considered as the gold standard when determining the performance of new microbial diagnostic methods. • Used to grow and multiply those bacteria which are suited to grow on the culture medium used which must include all necessary growth requirements.
  67. 67. DISADVANTAGES • Not all bacteria can be readily cultured . • Also, the use of selective media will restrict the species that are able to grow. • Strict sampling and transport conditions are essential. • Moreover, some of the putative pathogens, such as Treponema sp. And T.forsythus are fastidious and difficult to culture. • The sensitivity of culture methods is rather low, since the detection limits for selective and non-selective media average 103 to 104 bacteria and hence low numbers of a specific pathogen are undetected. • The most important drawback is that culture requires sophisticated equipment and experienced personnel and is relatively time-consuming and expensive.
  68. 68. DARK FIELD OR PHASE CONTRAST MICROSCOPY. • Darkfield or phase contrast microscopy can directly and rapidly assess the morphology and motility of bacteria in a plaque sample. • The main advantage of this technique is the ability to count all the bacteria in the sample. • These assays are inexpensive and take only a few minutes to perform.
  69. 69. DRAWBACKS Inability to differentiate the various species of microorganisms or determine their relative susceptibility to antimicrobial agents.
  70. 70. IMMUNODIAGNOSTIC METHODS • Employs antibodies that recognize specific bacterial antigens to dtect target microorganisms. • This reaction can be revealed using variety of procedures,including direct and indirect immunofluorescent microscopy assays(IFA), flow cytometry, ELISA, membrane assay and latex agglutination.
  71. 71. IMMUNOFLUORESCENCE ASSAY • Fluorescence is the property of certain dyes which absorb light in the ultraviolet region (200-400nm) and emit a characteristic wavelength of light (500-600nm).coons and Kaplan(1942) 1st introduced fluorescent labelled proteins.
  72. 72. IMMUNOLOGICAL ASSAYS DIRECT AND INDIRECT IMMUNOFLUORESCENT MICROSCOPY ASSAYS (IFA):
  73. 73. • IFA has been used mainly to detect A.a and P.g. • Zambon et al showed that this technique is comparable to culture in its ability to identify pathogens in subgingival plaque samples. IFA does not require viable bacterial cells. Comparative studies indicate that the sensitivity ranges from 82-100% for detection of A.a and 91- 100% for detection of P.g. with specificity values of 88-92% for A.a and 87-89% for P.g. • Drawbacks- separate fluorescent tagged antibodies has to be prepared against each antigen to be tested.
  74. 74. CYTOFLUOROGRAPHY OR FLOW CYTOMETRY
  75. 75. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA): • ELISA has been used primarily to detect serum antibodies to periodontopathogens and also in research to quantify specific pathogens in subgingival samples using specific monoclonal antibodies. The assay can only detect species for which a suitable antibody is available.
  76. 76. • The primary antibody is detected through a colorimetric reaction which is catalyzed through an enzyme (usually horseradish peroxidase or alkaline phosphatase) linked to the antibody. • The intensity of the colour depends on the concentration of the antigen and is read phometrically.
  77. 77. ADVANTAGES • Enzyme conjucated antibodies are stable and can be stored for relatively long time. • Formation of coloured product allows direct observation of the reaction. • Enzyme itself is not changes during activity, it can catalyse the reaction of many substrate molecules greatly ampliflying reaction.
  78. 78. LATEX AGGLUTINATION TEST • Latex beads are coated with the species specific antibody, and when these beads come in contact with microbial cell surface antigens or antigen extracts, cross-linking occurs and agglutination or clumping is seen within 2-5mins.
  79. 79. MERITS AND DEMERITS OF IMMUNOLOGICAL ASSAY • Immunological assays can identify dead target cells, thus not requiring stringent sampling and transport methodology. However, they cannot be used to determine antibiotic susceptibility. • Most of the assays provide a quantitative or semiquantitative estimate of target microorganisms.However, these methods generally show poorer detection limits than nucleic acid probes of PCR assays.
  80. 80. ENZYME BASED ASSAY BANA • It is an enzymatic assay used for the identification of the trypsin-like protease which is produced mainly by P.g and to a much lesser extent by T.forsythus and T. denticola and Capnocytophaga species. • This protease hydrolyses benzyol-DL-arginine-2- naphthylamide (BANA) substrates in a colorimetric assay. • The activity of the enzyme is measured with the hydrolysis of the colorless substrate BANA resulting in release of chromophore beta naphthylamide resulting in orange red colour.
  81. 81. • The main drawbacks are a lack of quantitative data and the inability to determine which of the three bacteria are responsible for enzyme production. • Also, the BANA system does not include inhibitors of host proteinases, which could cleave the BANA substrate and also contaminate the plaque sample from saliva and GCF.
  82. 82. • Loesche et al observed a close association with BANA positive sites (80-90%) with deep periodontal pockets (7 mm) • Beck et al used BANA test as a risk indicator for periodontal attachment loss.
  83. 83. PERIOSCAN (ORAL-B LABORATORIES) • Chairside test kit system which uses BANA test for trypsin-like proteinases. • A subgingival plaque sample is created in the kit with the substrate linked to a colour detection system which is particularly simple to use and their results are available within a relatively short period. They also produce visual results which can be shown to the patients.
  84. 84. DNA PROBES • DNA probe is a fragment of nucleic acid, labeled with an enzyme or a radioisotope, that can seek out and bind itself to other complementary sequences of DNA, thus forming the double helix structure found in nature.
  85. 85. • To prepare the probe, specific pathogens used as marker organisms are lysed to remove their DNA. Their double helix is denatured, creating single strands that are individually labeled with a radioactive isotope. Subsequently, when a plaque sample is sent for analysis, it undergoes lysis and denaturation. Single strands are chemically treated, attached to a special filter paper and then exposed to the DNA library. If complementary base pairs hybridize (cross-link), the radiolabeled strands will also be fixed to the filter paper. After the filter is washed to remove any unhybridized strands, it is covered with a radiographic plate. The radioactive labels create spots on the film which are read with a densitometer. The darkness and size of the spots indicate the concentration of the organisms present in the plaque sample.
  86. 86. TYPES (SAVITT ET AL 1990) • Whole genomic probes • Cloned probes • Oligonucleotide probes
  87. 87. WHOLE GENOMIC PROBES • These are constructed from the entire genome of the target microorganisms. They are easiest to construct and least expensive to produce. • Draw backs: similar species of the bacteria are likely to be present in the plaque samples hence cross reaction may occur.
  88. 88. CLONED PROBES • This is composed of isolated sequences of DNA that do not show cross reactivity. • Cloned probes can approach the sensitivity of whole genomic probe while avoiding cross reaction with other species (French et al 1986).
  89. 89. OLIGONUCLEOTIDE PROBES • These probes are 10-50 bp where as the other probe types are often several kilo base pairs in length. • These probes for oral species are relatively insensitive detecting only 106 cells compared to cloned and whole genomic probes.
  90. 90. OMNIGENE (OMNIGENE, INC.), DMDX TM TEST KIT (OMNIGENE, INC.) AND BTD (BIOTECHNICA DIAGNOSTICS, INC.): • These are DNA probe systems for a number of subgingival bacteria. A paper point sample of subgingival plaque is placed in the container provided and mailed to the company for assay. Probes are available for A.actinomycetemcomitans, P.gingivalis, P.intermedia, E.corredens, F.nucleatum, C.recta, T.denticola and T.pectinovorum.
  91. 91. • Luchnan et al 2003 used DNA probes for assessing the p.gingivalis showed DNA probe is more superior than culture (74% vs 20%).
  92. 92. PCR • By saiki and colleague in 1985. • PCR involves amplification of an exceedingly small region of DNA flanked by a selected primer specific for target species. • The presence of the specific amplification product indicates the presence of the target microorganisms. Among the different nucleic acid assays, PCR demonstrates the best detection limits, as few as 5-10 cells and shows no cross-reactivity under optimized amplification conditions
  93. 93. DETECTION OF PCR PRODUCTS • Specific pcr amplification products containing the target nucleic aid of interest is referred to as amplicon. • By radioactive, colorimetric, fluorometric or chemiluminescent signals.
  94. 94. MODIFICATIONS • Multiple PCR • Nested PCR • Quantitative PCR
  95. 95. • Mutiple PCR could detect as few as 50 Aa, p.g and 500 b.forsythus in plaque sample. • Compared to cultured methods PCR showed 45% sensitivity and 79% specificity fo A.a. • Ishikwa et al using PCR showed that the detection frequency of T.denticola and P.gingivalis in plaque samples from agg periodontitis is 73.7 and 84.2%; chronic is 93.8 and 95.3%; healthy subject has5 and 10% respectively.
  96. 96. DRAW BACKS • Relatively small aliquots are used for the amplification process. • So, if small quantity of plaque sample does not contain the target organisms the assay will not detect it. • Moreover, subgingival plaque may contain enzymes that can alter the amplification process.
  97. 97. ADVANTAGES OF DIAGNOSTIC TESTS USING BACTERIA AND THEIR PRODUCTS: • Some markers appear to be predictive of disease activity in longitudinal studies, e.g. GCF bacterial proteases. • The commercial tests are simple to use. • Results of chairside test kits are available in a short time. • Chairside test kits produce a visual result, which can be shown to the patient.
  98. 98. DISADVANTAGES OF DIAGNOSTIC TESTS USING BACTERIA AND THEIR PRODUCTS • The polymicrobial nature of the disease makes diagnosis difficult. • Most tests are not predictive of disease severity. • It is necessary to know which site to sample. • The tests only detect the bacteria that you look for. • Some need to be sent away to a special laboratory. • Cost.
  99. 99. HOST RESPONSE • Most of the diagnostic procedures/tests in periodontics have the limitation that they cannot differentiate between active and inactive sites at a given point of time, nor can they identify the susceptible individuals. • To overcome this difficulty, there have been concerted efforts in the last few years to study the components of GCF and blood or serum (less frequently).
  100. 100. • Inflammatory mediators and products. • Host derived enzymes • Tissue breakdown products
  101. 101. INFLAMMATORY MEDIATORS AND PRODUCTS • Cytokines are potent local mediators of inflammation present in GCF. • Cytokines investigated as potential markers include TNF-α, IL- 1 α, IL-1β, IL-6 and IL-8. IL-1, IL-6 and TNF-α are produced by macrophages and other cells at inflamed sites. • Good candidates for markers of disease progression.
  102. 102. • Prostaglandin E2 is formed as a result of the metabolism of arachidonic acid through cyclooxygenase pathway. It is a potent mediator of inflammation and bone resorption. In cases of active periodontal destruction, the level of GCF PGE2 is dramatically increased.
  103. 103. • Offenbacher et al demonstrated PGE2 levels can predict active periodontal disease progression.they studied 41 adult periodontitis patients over a 3 year period.
  104. 104. HOST DERIVED ENZYMES • Various enzymes are released from host cells during initiation and progression of periodontal disease. Some of these enzymes are released from dead and dying cells of the periodontium whereas some come from PMN and others are produced by inflammatory, epithelial and C.T cells at affected sites.
  105. 105. ASPARTATE AMINOTRANSFERASE (AST): • cytoplasmic enzyme • extracellular release is associated with cell damage and cell death. • marked elevation in AST levels in GCF samples from sites with severe gingival inflammation and sites with a recent history of progressive attachment loss. • Periogard is a rapid chair side test kit for AST • Drawback : Inability to discriminate between sites with severe inflammation but with no attachment loss from sites that are losing attachment. • Chambers et al demonstrated marked elevation in AST levels in GCF samples from site with severe gingival inflammation and sites with recent history of progressive attachment loss.
  106. 106. • Pocket watch: An in vitro diagnostic test kit to analyze AST levels in GCF and is used as an objective biochemical test for diagnosing and monitoring the disease activity to determine when to treat and also to evaluate the treatment effectiveness.
  107. 107. ELASTASE • produced by neutrophils in inactive form bound to inhibitor. • Active elastase is usually seen adjacent to JE where neutrophils are mirating. • Elastase is able to degrade proteoglycans and can also activate latent collagenase (Eley and Cox, 1990). • GCF elastase level significantly correlates with increasing gingival inflammation, probing depth, probing attachment level and bone loss and its level also significantly reduces following treatment (Eley and Cox, 1992)
  108. 108. PROGNOSTIK (DENTSPLY): • Detects the presence of the serine proteinase, elastase, in GCF samples.
  109. 109. ALKALINE PHOSPHATASE • Is found in many cells of the periodontium including osteoblasts, fibroblast and neutrophils. • Cross-sectional studies show that it is positively and significantly correlated with pocket depth but not with bone loss(Ishikawa and Cimasoni, 1970)and the concentration of this enzyme in GCF from diseased sites is significantly higher than that in healthy sites (Chapple et al .,1994) • Periocheck
  110. 110. Β-GLUCURONIDASE AND ARYLSULPHATASE • Both these enzymes are lysosomal and are found in the primary granules of neutrophils. • Lamster I B (1988) shown that concentration of this enzyme may have predictive value in identifying patients at higher risk for losing attachment.
  111. 111. COLLAGENASE: • Family of matrix metalloproteinases (MMPs) • synthesized by macrophages, neutrophils, fibroblasts and keratinocytes and are secreted as latent enzymes. • The 2 principal types of specific collagenase are MMP-8 which is found in inflammatory cells such as neutrophils and MMP-1 found in fibroblasts and other cells. • Collagenase (MMP-8, MMP-1) activity if found in gingival tissue, saliva and GCF and can be assayed biochemically with collagen substrates or by using monoclonal antibodies with ELISA.
  112. 112. • In human periodontitis, GCF collagenase activity has been shown to increase with increasing severity of gingival inflammation and increasing pocket depth and bone loss (Golub et al.,1996) • Total MMP-8 and MMP-8 concentration reduced significantly after treatment and this enzyme gave more significant correlations with clinical parameters and fell more after successful periodontal treatment than either elastase or cathepsin B (Chen et al.,1999).
  113. 113. TISSUE BREAKDOWN PRODUCTS • The tissues of the periodontium exist in a delicate balance between health and disease as well as between repair and regeneration. • Both catabolic and anabolic products from the ECM may be present in the GCF. These are potentially important marker of disease and tissue turnover (Embery et al 1991)
  114. 114. HYDROXYPROLINE • It is a amino acid of collagen and its appearance in GCF has been preliminarily investigated as a marker for the destruction of periodontal C.T .Analysis of GCF harvested from dogs during the development of experimental periodontitis showed elevated levels (Svanberg et al)
  115. 115. OSTEOCALCIN • 5.4 kD calcium-binding protein of bone. • Cross-sectional studies have shown that no significant amounts of osteocalcin is found in gingivitis, whereas varying levels are found in periodontitis. • The total amounts of GCF osteocalcin at diseased sites were significantly higher than those at healthy or gingivitis sites.
  116. 116. TO CONCLUDE CONDITION CLINICAL RADIGRAPHIC MICROBIAL HOST RESPONSE GINGIVITIS • BOP • GCF - - ASPARTATE AMINO TRANSFERASE CHRONIC PERIODONTITIS AUTOMATED PROBES • IOPA • OPG - ENZYMES PGE2,IL1,6,8, TNF-ALPHA AGG PERIODONTITIS - IOPA OPG DNA PROBE,PCR,I.F NECROTIZING - - DARK FIELD,DNA,ELISA,I .F - REFRACTORY - - CULTURING, I,F, DNA PROBE - REGENERATIVE THERAPY - DIGITAL RADIOGRAPHY,SU BTRACTION RADIOGRAPHY
  117. 117. REFERENCES • CARANZZA 10TH AND 11TH EDITION • LINDHE 4TH EDITION • MOLECULAR BIOLOGY OF PERIODONTIUM BY K.V.ARUN • Perio 2000 june 2016 • pubmed

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