Periodontal diagnostic tests include clinical methods, radiographic methods, microbial analysis, and host response analysis. Clinical methods include gingival bleeding, periodontal probing, tooth mobility, gingival temperature, and halitosis. Bleeding on probing and increased gingival temperature can indicate inflammation but are not reliable predictors of future attachment loss. Periodontal probing is important but multiple factors can affect accuracy. Digital radiography and subtraction radiography allow for enhanced detection and quantification of alveolar bone loss compared to conventional radiographs.

1. ADVANCED DIAGNOSTIC
AIDS IN PERIODONTICS
SHEETHALAN M S R

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. 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. PRINCIPLES OF DIAGNOSTIC TESTING
• Sensitivity
• Specificity
• Predictive value
• Positive
• Negative

5. PERIODONTAL DIAGNOSTIC TESTS
• Clinical methods
• Radiographic methods
• Microbial analysis
• Host response

6. CLINICAL METHODS
• Gingival bleeding
• Periodontal probing
• Tooth mobility
• Gingival temperature
• Halitosis

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. • 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. • 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. 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. • 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. 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. 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. 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. 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. 1ST GENERATION

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. 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. • 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. • 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. 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. 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. 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. 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. 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. • 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. 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. 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. PROBES FOR FURCATION INVOVLEMENT
• Naubers probe

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. EVIDENCES

34. 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.

35. 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)

36. 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.

37. • -8 to +9 : clinically firm tooth
• 10-19 : palpable mobility
• 20-29 : visible mobility
• 30-50 : mobility in response to lip and tongue movements

38. • Goodson (1988) confirmed the correlation between PTV and
clinical mobility index (MI).
• The greater the alveolar bone height, the lower the periotest
value.

40. 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.

41. • ORAGANOLEPTIC RATING
• GAS CHROMATOGRAPHY
• DARK FIELD MICROSCOPY
• SALIVA INCUBATION

42. • 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.

43. • 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.

44. 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.

45. • 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.

46. LIMITATION
• Only 2 dimentional images.
• Errors due to exposure and processing.

47. 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

48. 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.

49. • 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

50. 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

51. 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.

52. • 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.

53. 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.

54. 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.

55. • 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.

56. 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.

57. 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

58. • 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.

60. 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.

61. • 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.

62. 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.

63. • 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.

64. 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.

65. • 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.

66. • 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.

67. 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.

68. 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.

69. • Culture techniques
• Dark ground or phase contrast microscopy
• Immunological assays
• DNA Probes
• Enzyme based assays

70. 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.

71. 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.

72. 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.

73. DRAWBACKS
Inability to differentiate the various species of microorganisms or
determine their relative susceptibility to antimicrobial agents.

74. 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.

75. 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.

76. IMMUNOLOGICAL ASSAYS
DIRECT AND INDIRECT IMMUNOFLUORESCENT MICROSCOPY ASSAYS (IFA):

77. • 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.

78. CYTOFLUOROGRAPHY OR FLOW
CYTOMETRY

79. 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.

80. • 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.

82. 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.

83. 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.

85. 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.

86. 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.

87. • 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.

88. • 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.

89. 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.

90. 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.

91. • 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.

92. TYPES (SAVITT ET AL 1990)
• Whole genomic probes
• Cloned probes
• Oligonucleotide probes

93. 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.

94. 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).

95. 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.

96. 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.

97. • Luchnan et al 2003 used DNA probes for assessing the
p.gingivalis showed DNA probe is more superior than culture
(74% vs 20%).

98. 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

100. 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.

101. MODIFICATIONS
• Multiple PCR
• Nested PCR
• Quantitative PCR

102. • 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.

103. 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.

104. 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.

105. 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.

106. 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).

107. • Inflammatory mediators and products.
• Host derived enzymes
• Tissue breakdown products

108. 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.

109. • 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.

110. • Offenbacher et al demonstrated PGE2 levels can predict active
periodontal disease progression.they studied 41 adult
periodontitis patients over a 3 year period.

111. 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.

112. 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.

113. • 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.

114. 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)

115. PROGNOSTIK (DENTSPLY):
• Detects the presence of the serine proteinase, elastase, in GCF
samples.

116. 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

117. 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.

118. 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.

119. • 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).

120. 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)

121. 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)

122. 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.

123. 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

124. REFERENCES
• CARANZZA 10TH AND 11TH EDITION
• LINDHE 4TH EDITION
• MOLECULAR BIOLOGY OF PERIODONTIUM BY K.V.ARUN
• Perio 2000 june 2016
• pubmed