Efficacy of Diagnostic Techniques Flashcards
Microbiologic Testing
Expensive and well-training personnel required Bacterial culturing Direct microscopy Immunodiagnostic methods Enzymatic methods Molecular biology techniques
Assessment of the Host Response
Biochemical analysis as part of periodontal diagnosis
Source of samples:
GCF, saliva, and serum (blood)
Genetic Analysis (only for research)
There is a genetic susceptibility to periodontitis
Gene polymorphism as a risk marker for periodontitis
Probe Penetration
Lack of sensitivity and reproducibility
Probing depth: gingival inflammation, insertion force, placement and angulation, size, probing technique, probe calibration, presence of subgingival calculus, overhanging restorations
CAL
Poor reliability and reproducibility
Limited practical value
Radiographic Examination
Limited sensitivity in small bone change
– Changes in bone can be identified by eye only after 30% to 50% of the bone mineral has been lost (Subtraction
radiography: detect bone density change as low as 5 %)
No value in evaluating disease activity or progression
Radiographic exam: simply a measurement of the
past damage of a disease process. +- 1mm for probing depth – this will vary based on person to person.
Ultrasonic periodontal probe uses a
hollow tapered tip that is filled with water for coupling of the ultrasonic beam into the tissues (non-invasive)
Cone-beam Computed Tomography: Conventional radiographs (PA; Pano) are very
specific, but lack sensitivity
Cone-beam Computed Tomography: Recently, dental CBCT has been introduced in periodontology for the detection of
periodontal defects in in vitro settings
Cone-beam Computed Tomography: CBCT is promising for periodontal applications, especially for
intrabony defects, dehiscence and fenestration defects, periodontal cysts, furcation defects and thickness of palatal masticatory mucosa
The sensitivity of a diagnostic test refers to the
probability of the test being positive when the disease is truly present
The specificity of a diagnostic test refers to the
probability of the test being negative when the disease is not present
Fastidious microorganism – microorganisms that will grow only if
special nutrients are presence
Assess for antibiotic susceptibility of microbes while
culturing
Some putative pathogens are fastidious and
difficult to culture
Sensitivity is low: detection limits for selective and nonselective media average
104 to 105 bacteria
- Direct Microscopy
Alternative to culture methods
Dark-field or phase-contrast microscopy
Morphology and motility of bacteria in a plaque sample
Problem with direct microscopy:
Most of the main putative perio pathogens are non-motile (so it is difficult to identify)
- Immunodiagnostic Methods
Use Ab that targets specific
bacterial Ag
Direct and indirect - immunofluorescence microscopic assay
immunofluorescent microscopic assay (IFA)
Able to identify pathogens using a plaque smear Used mainly to detect Aa and Pg Comparable to bacterial culture Does not require viable bacterial cells
- Immunodiagnostic Methods: Cytofluorography (flow cytometry)
complexity and cost prevent its wide use
Immunodiagnostic Methods: Enzyme-linked immunosorbent assay (ELISA)
Used primarily to detect serum antibodies to periodontal pathogens Membrane immunoassay (EvalusiteTM): chairside use to detect Aa, Pg, and Pi (detection limit of 105 for Aa and 106 for Pg)
Immunodiagnostic Methods: Latex agglutination
Based on the binding of protein to latex: latex beads are coated with species-specific antibody
Currently these assays only for research purposes
- Enzymatic Methods
Several putative periodontal pathogens such as Pg, Tf, and Aa possess in common a trypsin-like enzyme that hydrolyzes a substrate N-benzoyl-DL-arginine-2-naphthylamide (BANA).
Chair-side kit (PerioscanTM) was available in the 1990s
Inability to distinguish between individual bacteria
- It may be positive at clinically healthy sites
Negative result does not rule out the presence of other important periodontal pathogens
16s rRNA is like a finger print:
comparing certain locations on a 16s rRNA molecule with a database of known organisms allows the identification of organisms whose 16s rRNA signature is known.
Real-time PCR is also known as
The quantitative real time polymerase chain reaction (qPCR). The Real-Time-PCR enables both detection and quantification.
Nucleic acid probes
Synthesized and labeled DNA (20-30 nucleotides)
Genomic DNA probe (whole DNA strand): significantly ↓ in sensitivity and specificity due to cross-reactivity to non-target microorganisms
16S rRNA – oligonucleotide probes (high sensitivity and specificity
Checkerboard DNA-DNA hybridization
Whole genomic digoxigenin-labeled DNA
Up to 40 oral bacterial species in a single test
Not been generalized for diagnostic purpose
PCR
High sensitivity and specificity for the identification of target pathogens
PCR lower detection limit: 25-100 cells (Culture: 104-105 cells)
Unable to quantify pathogens accurately in clinical samples
Real-time PCR
Real-time PCR: good correlation between the fluorescent signal measured and the number of bacterial cells been used
Expensive and sophisticated technology in real-time PCR
Hypophosphatasia (OMIM 146300, 241500, 241510) is an inherited disorder characterized by
defective bone and teeth mineralization and deficiency of serum and bone alkaline phosphatase (AP) activity.
urinary phosphoethanolamine (PEA) is increased in
hypophosphatasia.
urinary inorganic pyrophosphate (PPi) level is
also increased in hypophosphatasia
Collecting GCF
Paper strips placed within the crevice for 30 seconds
Fluid volume can be quantified by Periotron®
Captured samples may not represent the entire periodontium
Selection of the teeth and sites is often difficult
GCF
Currently > 65
components of GCF have been evaluated
Host-derived enzymes and their inhibitors
Byproducts of tissue breakdown
Inflammatory mediators and host-response modifiers
Intracellular destruction enzymes
Possible markers of active periodontal destruction
Released from dead or dying PMN/Neutrophils from periodontium
Aspartate amino-transferase: released during
tissue destruction (cell death)
- Alkaline phosphatase: a membrane-bound
glycoprotein involved in maintenance of alveolar bone
- β-glucuronidase: a
lysosomal enzyme degrades proteoglycans and ground substance
- Elastase: a
proteolytic enzyme found in lysosomal granules of neutrophil
Extracellular destruction enzymes
Associated with the activity of matrix metalloproteinases
Produced by inflammatory, epithelial and connective tissue cells
Aspartate aminotransferase (AST)
Periogard Periodontal Tissue MonitorsTM (chair-side test kit)
A marked elevation in AST levels in GCF from sites with severe gingival inflammation
Inability to discriminate between sites with severe inflammation with or without attachment loss
Alkaline phosphatase (ALP)
ALP in GCF are higher in diseased then healthy sites alkaline phosphate in disease.
β-glucuronidase (βG)
Elevated βG in GCF from sites with severe periodontal disease
High sensitivity and specificity when related to occurrence of clinical attachment loss
Good predictor for future periodontal breakdown
Elastase
Periocheck® (chair-side test kit)
Positive correlation of elastase in GCF with clinical attachment loss
Matrix metalloproteinases (MMPs) Secreted by
fibroblasts and macrophages
MMPSL: Responsible for
remodeling and degradation of ECM components
MMPS: Regulated by
tissue inhibitors of MMPs (TIMPs)
periodontal ECM
MMPS: High MMP levels in GCF are at significantly greater risk for
progression of periodontitis
GCF MMPs level reduces in response to
treatment
Destruction of collagen
The ECM of the periodontium is composed of
collagen (predominant), proteoglycan (versican, decorin, biglycan, syndecan) and non-collagen proteins (elastin, fibronectin, laminin, osteocalcin, osteopontin, bone sialoprotein, osteonectin, and tenascin)
Elevated levels of hydroxyproline (breakdown from collagen), glycosaminoglycans (from matrix degradation) and osteocalcin & type I collagen (from alveolar bone destruction) can be found in the
GCF from sites with periodontitis
Traditional immunoassays analyze only a
single cytokine at a time
Cytokines
inflammatory mediator
Bio-Plex Cytokine Assay
Incorporates novel technology using color-coded beads, permits the simultaneous detection of up to 100 cytokines in a single well of a 96-well microplate
Are multiplex bead-based assays designed to quantitate multiple cytokines in tissue fluid including GCF
OPTICAL SPECTROSCOPY
Infrared (IR) Spectroscopy
Vibrating covalent bonds of organic molecules absorb a characteristic wavelength of IR light
The wavelength of light absorbed depends on the nature of the covalent bond (e.g., C=O and N–H), the type of vibration (e.g., bending and stretching), and the environment of the bond
The spectrum of absorbed light can be used to establish a molecular fingerprint of a tissue or fluid
Diagnosis of Periodontitis Based on IR Spectra of GCF
Measure the total contents of GCF
IR spectroscopy can be used to characterized GCF from healthy, gingivitis, and periodontitis sites
Vibrations of peptide groups: C=O stretching (amide I band) and N–H bending (amide II band)
IR spectroscopy of GCF is reagent free, requiring only small sample volumes, requiring minimal training for operator
Near Infrared (NIR) Spectroscopy
Measure of oxygen saturation of the tissues
The wavelength region 500 to 600 nm is dominated by the absorption from oxygenated hemoglobin (HbO2) and deoxygenated hemoglobin (Hb)
Tissue oxygenation at periodontitis sites significantly ↓ as compared to gingivitis and healthy control sites
Why Saliva for periodontal disease testing?
Abundant fluid and easy to collect and store
Highly enriched content of disease biomarkers
- Such as Sjogren’s syndrome and oral cancer
Salivary Occult Blood Test (SOBT) –
available in Japan
A poor periodontal status: Subjects having ≥15% of teeth with BOP or ≥ 1 tooth with PD ≥ 4mm
A paper strip containing gold-labeled anti-human hemoglobin monoclonal antibody was dipped into saliva sample (Positive: ≥ 2 μg/ml human hemoglobin)
Sensitivity:0.72; Specificity: 0.52
The SOBT may offer a simple screening method for periodontal status when
a thorough periodontal examination is not possible, although it is not sufficiently specific to be a reasonable substitute for a periodontal examination
