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