Advanced Diagnostic Aids in Dentistry

Contents Introduction Limitations of conventional periodontal diagnosis Advances In Clinical Diagnosis Advances In Radiographic Assessment Advances Microbiologic Analysis Advances In Characterizing The Host Response

Introduction Definition : Diagnosis is defined as identifying the disease from an evaluation of history, signs and symptoms, laboratory tests and procedures. Importance : It identifies and indicates the nature of etiological factors Indicates the nature of pathological processes It is essential for treatment planning

types of diagnosis Provisional differential comprehensive therapeutic emergency Diagnostic aids in periodontics conventional advanced

Periodontal disease Gingival Inflammation Periodontal Destruction Diagnosis :- clinical evaluation ( Gingivitis) clinical evaluation + (periodontitis) tissue destruction

Tissue destruction ( Periodontitis) Loss of connective tissue clinical radiographic attatchment loss bone loss Gives historical evidence of damage Identify and quantify current clinical signs of inflammation

Limitations Does not provide cause of condition Susceptibility of patient Cannot reliably identify sites with ongoing periodontal destruction Cannot differentiate whether response to therapy is positive or negative

Periodontal disease is localised and multifactorial.

Consideration should be given microbiologic immunologic systemic genetic behavioural factors in addition to clinical and radiographic parameters.

The focus is now disease prevention, early discovery, and intervention to minimize treatment, thus enabling the most desirable outcomes. Diagnostic modalities available to clinicians today expand greatly on the foundation of a comprehensive visual assessment, which has been and will be the cornerstone of the diagnostic process.

Classification Aids used in clinical diagnosis Conventional probes – regular examination Millimeter probes – for gingival bleeding Pressure sensitive probes Other clinical diagnostic aids Filter papers- for measuring GCF Periotron 6000- for measuring GCF Olfactometer- for mouth odors Mobilometer- for tooth mobility PSR- for faster screening and recording of PD

Aids used in radiographic diagnosis IOPA radiographs Ortho-pantomograph Xero-radiography Aids used in microbial diagnosis Direct examination Light microscopy Dark field microscopy Culture tests Aerobic culture Anaerobic culture

Aids used in immunological diagnosis Immunofluorescense- direct and indirect Polymerase chain reaction Latex agglutination Flow cytometry ELISA Biochemical diagnosis Studies for prostaglandins Studies for collagenase Other diagnostic aids i. study casts ii. FSEIA iii. N-benzoyl-DL- arginine 2- naphthylamide (BANA)

Advanced diagnostic aids Advanced periodontal probes Automated controlled force probes Thermal periodontal probes Advanced diagnostic aids in periodontal radiography Digital radiography Substraction radiography Digital substraction radiography Transmission radiography Magnetic resonance imaging Computerized tomography Nuclear medicine bone scan

Advanced diagnostic aids in microbiologic analysis Advances in culturing technique Advances in immunodiagnostic methods Advances in enzymatic methods Advances in nuclear biology- PCR and DNA probes Advanced diagnostic aids in charting the host response Assessment of inflammatory mediators and products Assessment of tissue breakdown products Assessment of host derived enzymes

Advanced diagnostic aids to determine periodontal disease activity Crevicular contents Products of bacteria Products of host cells and host immunity Markers in peripheral blood Neutrophil functional profile Monocyte responsiveness to LPS Circulating antibodies to plaque bacteria Detection of specific periodontal pocket bacteria a. DNA probes b. BANA hydrolysis c. Antibody techniques

Advances in clinical diagnosis Degree of gingival inflammation a. redness and swelling b. gingival bleeding Gingival bleeding ∞ plaque accumulation gingival inflammation (Greenstein G et al 1981) size of inflammatory infiltrate probability of losing attatchments (Lang et al.1991)

Gingival temperature Thermal probes are sensitive diagnostic devices for measuring early inflammatory changes in gingival tissue. (Kung et al 1990) Commercially available system periotemp probe Individual temperature differences are compared with those expected for each tooth and higher temperature pockets are signaled with a red emitting diode. Subgingival temperature at diseased sites is increased as compared to normal healthy sites

Elevated subgingival temperature ∞ attatchment loss & elevated propertions of periodontopathic bacteria ( Haffajee et al.)

Periodontal probes Most widely used Clinical assessment of connective tissue destruction in periodontitis Gold standard – recording changes in periodontal status Probing depth is measured from the free gingival margin (FGM) to the depth of the probable crevice. not the most objective measure of loss of periodontal tissues

CAL is a more objective measure of loss of Existing periodontal support. CAL also does not give any indication of current disease activity. When interpreting the PD and CAL measurements made with conventional periodontal probes, it is important to consider that these values depend on the inflammatory state of the tissues.

Classification of periodontal probes depending on generation. 1.First generation probes:(conventional probes) Conventional manual probes that do not control probing force or pressure and that are not suited for automatic data collection. eg: Williams periodontal probe CPITN probe UNC-15 probe University of Michigan’O’ probe Goldman Fox probe Glickman probe Merritt A and B probe

2.Second generation probe: (Constant force probe) Introduction of constant force or pressure sensitive probes allowed for improved standardization of probing. e.g.: Pressure sensitive probe Constant pressure probe 3.Third generation probe:(Automated probes) Computer assisted direct data capture was an important step in reducing examiner bias and also allowed for generation probe precision. e.g.: Toronto probe Florida probe Interprobe, Foster Miller probe.

4.Fourth generation probes:(Three dimensional probes) Currently under development, these are aimed at recording sequential probe positions along a gingival sulcus. An attempt to extend linear probing in a serial manner to take account of the continuous and three dimensional pocket that is being examined. 5.Fifth generation probe:(Noninvasive) Three dimensional probe. Basically these will add an ultrasound to a fourth generation probes. If the fourth generation can be made, it will aim in addition to identify the attachment level without penetrating it. e.g.: Ultra sonographic probe.

Florida probe: Tip is 0.4mm Sleeve- edge provides reference to make measurements Coil Spring; provides constant probing force Computer for data storage.

Disadvantages of Florida probe. Lack of tactile sensitivity Fixed probing force Underestimation of deep periodontal pockets. Other electronic probes: Improvised Florida PASHA probe Interprobe Perioprobe Foster Miller probe Toronto Automated ( difficult to reproduce patient head position and in 2nd and 3rd Molar area.)

PSR system To screen dental patients to facilitate the detection of mild forms of periodontal diseases and to identify individuals who have previously undetected periodontitis. It is designed for general dental practitioner to identify the patient’s requiring periodontal care and to determine the type of care required.

Based on the worst site per sextant. If one or more sextants show significant signs of disease, clinician is advised to do a complete periodontal examination and charting

Advances in Radiographic Assessment Dental Radiography are traditional method to assess destruction of alveolar bone. Problems with conventional Radiography: Variation in projection geometry Variation in contrast and density Masking by other anatomic structures. They are very specific but lack sensitivity.

Digital Radiography Computerized images. Image property almost equal to conventional radiographs 1/3rd to half reductions in radiation dose.

Substraction Radiography Well established in medicine Introduced in Periodontal diagnosis Principle: Serial radiographs converted to digital images superimposed composite image– Quantitative changes Changes in density and volume of bone a. can be detected as lighter areas (bone gain) b. Dark areas (bone loss)

Perfect accuracy at a lesion depth of 0.49 mm ( Grondhal et al 1988) Limitations: needs paralleling technique and accurate superimposition.

Diagnostic Subtraction Radiography Positioning device during film exposure specialized software designed for digital image subtraction using conventional personal computers Applies an algorithm that corrects for the effects of angular alignment discrepencies and provides some degree of flexibility in imaging procedure

Computer Assisted Densitometric Image Analysis (CADIA) Video cameras measures light transmitted through Radiographs signals from camera converted into gray scale images camera interfaced with image processor + computer

Offers objective method for following alveolar bone density changes quantitatively over time. Higher sensitivity High degree of reproducibility accuracy

COMPUTED TOMOGRAPHY Computed Tomography scanning is widely used in the evaluation of the implant patient

A thin fan beam of X-Rays rotates around the patient to generate in one resolution a thin axial slice of the area of interest. Multiple overlapping axial slices are obtained by several revolution of the X-ray beam until the whole area of interest is covered. With the help of a computer and sophisticated Algorithms these slices are then used to generate a three dimensional digital map of the jaw which help in evaluation of the implant patient.

Specialized software can be used to generate appropriate views that best depict the dimensions of the jaws and the location of important anatomic structures. DENTAL VIEWS OBTAINED FROM A CT SCAN INCLUDE:- 1. AXIAL 2. PANORAMIC 3. CROSS-SECTIONAL. Views of the Jaws.

ADVANTAGES of Computed Tomography 1. True cross sections offer a precise and detailed evaluation of the height and width of the ALVEOLAR RIDGE. 2. The images can be adjusted and printed without magnification, facilitating measurements directly on the prints or films with standard rulers. 3. Various anatomic structures can be visualized and analyzed at all the Coordinate axis. 1. Superioinferior 2. Anteroposterior 3. Buccolingual

Images of the entire arch several edentulous areas can be visualized with single examination. The Bone and soft tissue contrast and resolution are excellent for the diagnostic task.

DISADVANTAGES of Computed Tomography specialized equipment and setting. Radiologists and Technicians need to be knowledgeable of the anatomy, anatomic variants and pathology of the jaws as well as considerations pertinent implant treatment planning. higher radiation dose to the patient as compared to other modalities used during implant treatment planning. it delivers radiation to whole arch. Metallic Restorations can cause ring artifacts that impair the diagnostic quality of the image, it is challenging to the patients having heavy metallic restored dentition.

CONE-BEAM COMPUTED TOMOGRAPHY Cone-Beam Computed Tomography (CBCT) is a new imaging modality that offers significant advantages for the evaluation of implant patients.

The 3- D CBCT images canbe combined with high precision 3D visible light (photographic) surface images to create a virtual patient that accurately displays both hard and soft tissue structures. This multi- modal image image visualization enables a treatment platform that allows assessment of the patient’s present condition, planning and stimulation of treatment options, progress monitoring and evaluation of outcomes.

Advances in Microbiologic Analysis Strong evidence for actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Tannerella forsythia (Tf). Other organisms that are thought to have etiologic role are Camphylobacter rectus, Eubaterium nodatum, Fusobacterium nucleatum, Peptostreptococcus micros, Prevetolla intermedia and Prevetolla nigrescens, Trepenoma Denticola

Microbiologic tests( that can identify) Can support diagnosis of various Periodontal disease. Can tell about initiation & progression Active & Inactive Can also be used to monitor Periodontal therapy whether it is suppressed/ eradicated. Several studies (-)nce of Pathogens better periodontal health (+)nce of pathogens periodontal disease

Bacterial culturing Direct microscopy Immunodiagnostic methods Enzymatic methods Diagnostic assays based on molecular biologic techniques

Bacterial culturing Historically, widely used in characterizing composition of subgingival microflora. Plaque sample anaerobic culture selective non-selective Advantage: 1. clinician can obtain absolute or relative count 2. invitro method for antibiotic susceptibility

Limitations : Culture can grow only live bacteria. Sensitivity is low Requires sophisticated equipment and experienced personnel. Time consuming expensive

Direct microscopy Dark field Direct microscopy alternative to culture methods Ability to assess morphology & motility in plaque. Dark field microscopy seems an unlikely candidate as a diagnostic test of destructive periodontal diseases.

Immunodiagnostic methods It employs Antibody that recognize specific bacterial antigen Various procedures: Direct & indirect immunofluorescence assays Flow cytometry ELISA Membrane assay Latex agglutination

Immunofluorescence assay Direct IFA: AB conjugated with Fluorescein marker + Bacteria ( Antigen) = Immuno complex Indirect IFA: Primary AB + Bacteria= Immune Complex+ Secondary Fl conjugated AB

Indirect IFA Direct IFA

Cytofluorography/ Flow cytometry Bacterial cells+ species specific AB + Secondary FL Conjugated AB Introduced in flowcytometer Bacterial cells is separated into single cell suspension-  passes through the tube Cells identified by lasers

merits : rapid identification labels bacterial cells species specific antibody fluorescin conjugated secondary antibody Demerits: sophistication expensive

ELISA= Enzyme Linked Immunosorbent Assay Similar AB and Antigen reaction, but the fluorescence is read using a photometer. Evalusite: commercially available kit to detect Aa,Pg and Pi.

Specific antigen bind to the antibody + Secondary antibody added.  Well with precoated antibody + Sample to be tested= immune complex Specific antigen bind to the antibody + Secondary antibody added. Immunofloresence dye bound to secondary antibody  Substrate added which changes the color of the solution Amount of florescence checked by photometer (450nm)

Latex Agglutination Test Latex beads coated with species specific AB when beads come in contact with specific species in sample they bind and agglutination occurs clumping of beads is visible test positive. Advantages: Simple and Rapid testing Higher sensitivity and specificity.

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. Enyme hydrolyses specific substrate (BANA) release cromophore ( B-naphtalamide) Cromophore changes color on addition of a substance( fast garnet)

Perioscan is a popular diagnostic kit uses BANA reaction. Disadvantage: May be positive in clinically healthy site Cannot detect disease activity Limited organisms detected Other pathogens may be present if it’s negative.

Molecular Biology Techniques Basic Principle: Analysis of DNA, RNA and protein structure. Hybridization: Pairing of complimentary strands of DNA to produce a double stranded DNA . Nucleic acid probe: is a known DNA/RNA which is synthesized artificially and labeled with a enzyme or a radioisotope for detection when placed in a plaque sample

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.

DNA Probe Whole genomic: 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.

Oligonucleotide probes: target variable region of 16sRNA or a specific sequence in the DNA strand. Higher sensitivity and specificity.

Checkerboard DNA-DNA Hybridization Technology: Developed by Socransky et.al. 40 bacterial species can be detected using whole genomic digoxigenin-labeled DNA probes. Large number of samples can be tested and upto 40 oral species detected with a single test.

Advantages of DNA probes as compared to bacterial culturing. More sensitive and specific Requires as less as 104 cells of each species to be detected. Multiple species detected with a single test Does not require viable bacteria Large number of samples can be assessed. Disadvantage: Expensive Expert personnel to carry out the test Not easily available

Polymerase Chain Reaction (PCR): 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.

Advantages: High detection limit. As less as 5- 10 cells can be amplified and detected. Less cross reactivity under optimal conditions Many species can be detected simultaneously Disadvantage: Small quantity needed for reaction may not contain the necessary target DNA Plaque may contain enzymes which may inhibit these reactions.

Advances in characterizing the host response Diagnostic tests have been developed that add measures of the inflammatory process to conventional clinical measures. These diagnostic tests gives information about pattern of destruction current activity of disease rate of disease progression extent & severity of further breakdown response to therapy

So, knowing about disease destructive process- clinician can individualise their therapeutic approach, and customize the treatment. Source of samples may be; GCF, Saliva, or Blood. GCF is most commonly used, where as saliva is recently been researched. Assessment of Host response Inflammatory mediators and products Host derived enzymes Tissue breakdown products

Inflammatory mediators & products: Cytokines- potent local mediators of inflammation produced by variety of cells GCF- TNF α IL-1 IL-6 IL-8 Good correlation with disease status and severity but not disease progression

In untreated periodontitis- concentration of PGE2 increased during active phase of periodontal destruction. Cytokines ∞ disease severity & status PGE2 ∞ active phase of periodontal disease

Host derived enzymes: Matrix components are dissolved by either ECM metalloproteinase dependent Plasmin dependent cleavage reactions Proteases and enzymes involved in these processes may be used as diagnostic aids.

Aspartate aminotransferases Collagenase β-glucuronidase Lactate dehydrogenase Arylsulfatase Elastase Alkaline phosphatase

Tissue Breakdown Products Hydroxyproline Glycosaminoglycans

For periodontal diagnosis, the ideal diagnostic test should be Quantitative. Highly sensitive method capable of analyzing a single periodontal site in health as well as disease. Reproducible. Highly specific. Simple to perform. A rapid, one or two stage procedure. Non-invasive. Versatile in terms of sample handling, storage and transport. Amendable to chairside use. Economical. ( Chapple 1997)

Chairside periodontal test kits can be categorized as Microbiologic tests Biochemical test kits Genetic kits Microbiological test kits The bacteriological tests (Microscopy, Culture, Omnigene, Affirm DP and Evalusite) are mainly aimed at spirochetes, Aa, Pg and Pi. Microbial tests can also be used to monitor periodontal therapy directed towards the suppression or eradication of periodontopathogenic  microorganisms

Omnigene DNA probe systems available for the detection of A. actinomycetemcomitans, P. gingivalis, P. intermedia, F. nucleatum, C. rectus, T. denticola and E. corrodens.

Evalusite Evalusite is a kit that employs a novel membrane-based enzyme immunoassay for the detection of three putative periodontopathogens: Aa, Pg and Pi.  a pink spot is displayed if the test organism is present. The main weaknesses of this test kit reside in 1) the assumption that the three detected organisms are causing disease; (2) it is a multistage test; (3) it has a subjective calorimetric end point and (4) there is no permanent record of the results.

PerioScan® Perioscan is a diagnostic test kit that utilizes the BANA (N-benzoyl-DL-arginine-2-naphthylamide)-hydrolysis reaction, developed to detect bacterial trypsin-like proteases in the dental plaque

Biochemical test kits Perio 2000- to evaluate VSCs Prognos- Stik- to detect elevated levels of MMPs Perio- Check- to measure neutral protease activity PerioGard- to detect AST Pocket Watch- simple method for AST

Genetic test kits Various gene polymorphisms are considered to be risk factors for the initiation or progression of periodontal disease. In 1997, Kornman et al. found an association between the polymorphism in the genes encoding for interleukin-1α and interleukin-1β and increased severity of periodontitis. PST® genetic susceptibility test - the first and only genetic test that analyzes two interleukins (IL-1α and IL-1β) genes for variations. IL-1 genetic susceptibility may not initiate or cause the disease but rather may lead to earlier or more severe disease

Salivary diagnostics   OralDNA® Labs has developed a salivary test, the MyPerioID® PST®, that identifies a patient’s genetic susceptibility and inherent risk to periodontal disease by evaluating their interleukin-1 (IL-1) gene cluster, MyPerioPath®, that identifies the type and concentration of 13 pathogenic bacteria known to cause periodontal disease.

Conclusion In periodontology, the success of any treatment is dependent upon the accuracy of the initial diagnosis. At present, the majority of chronic periodontitis cases can be adequately managed using existing diagnostic methodology, although it is clearly more desirable to be able to diagnose “active disease” as it occurs, rather than months later.   However, the clinician must ensure that the use of such tests will benefit the patients in terms of both the value of diagnostic data obtained and the cost in time and money. 

References Carranza’s Clinical Periodontology 10th and 11th edition. Armitage GC, Jeffcoat MK, Chadwick DE: longitudinal evaluation of elastase as a marker for the progression of Periodontitis, J Periodontol 1994; 65:120. Beck JD: Issues in assessment of diagnostic tests and risk for Periodontal diseases, Periodontol 2000;7:100

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