ADVANCED MICROBIAL DIAGNOSTIC TECHNIQUES IN

PERIODONTICS

Dr. Sarita Joshi Narayan, Dr. Umesh Yadalam, Dr. Vijay Raghava, Dr. Neha

Korla, Dr. Nithika Jain

ABSTRACT Based on the specific plaque theory in

periodontology, several chairside tests have been introduced for the microbiological examination of periodontal patients.

For the majority of these patients, however, a standard therapy (root planing in combination with good oral hygiene) results in a favorable outcome. Therefore, only for a small minority of periodontal patients a microbiological analysis could possibly contribute to better treatment.

For such patients, a choice made between a culture technique(which is labour intensive and has a long waiting time before an answer is available) and one of the chairside tests.

The culture technique has the advantage to examine most of the flora.

Major challenges in periodontology include understanding the pathophysiology, the interplay between various components of the host response, parallels with other diseases and identifying biomarkers of the disease.

INTRODUCTION Gram negative anaerobes are the major microorganism of

chronic and aggressive periodontitis. (Dzink et al, 1985 & Neumann, 1990)

Evidence of etiology is based on Socransky’s criteria.

Various organisms responsible for causing chronic and aggresssive periodontitis are:

Aggregatibacter actinomycetemcomitans, Porphromonas gingivalis, Tannerella forsythus, Camplyobacter rectus, Peptostretococcus micros and Prevotella intermedia

A few microbiological tests are now available including:

1. Direct microscopy- phase contrast, dark field2. Virulence factor- enzymes( Bana test)3. Specific analyses- culture and senstivity4. Immunologic assays- immunofluoresence,

enzyme immunoassay5. DNA/ oligonucleotide probes

BACTERIAL CULTURING Gold standard for microbial testing, when

determining the performance of new microbial diagnostic methods.

It helps in diagnosing infectious diseases and allows identification of established periodontal pathogens in particular.

In addition bacterias such as pseudomonas, enteric rods, staphylococci, candida species, etc. can be identified.

Clinician can obtain relative and absolute counts of the cultured species.

This is the only in vitro method able to assess for antibiotic susceptibility of the cultured organisms.

Short-comings:1. Although, non-selective media can be used

in such assays, it is recognized that not all organisms present in plaque sample will grow equally well, so that the results from the culture may not reflect what is present in situ.

2. Also, some putative pathogens like Treponema species are fastidious and difficult to grow.

3. Sensitivity of culture methods is low.4. This type of test requires specially anaerobic

laboratory, experienced personnel and is relatively time consuming and expensive.

DIRECT MICROSCOPY Evaluation of the subgingival infection can

involve assessment of the shapes and motility of the organisms in the subgingival plaque.

This can be accomplished with either phase-contrast or more commonly, dark field microscopic examination.

The dark field characteristics of the subgingival microflora were evaluated by Listgarten and Hellden(1978)

The presence of spirochetal forms, as well as other organism that are motile under dark field illumination has been associated with existing periodontal disease.

Although, the relationship of dark field morphotypes to the progression of periodontal disease has been investigated in several studies, the results have been inconclusive.

Significant disadvantages of dark-field microscopy are:

1. Absence of species identification2. Lack of guidance concerning the choice of

appropriate antibiotic.(Listgarten,1992)Dark-field microscopy is no longer being

extensively studied as a diagnostic test for periodontal disease.

PHASE- CONTRAST MICROSCOPYAdvantages:1. It can detect a number of high risk bacterial

morphotypes including spirochetes and motile cells.

2. It can also detect WBCs, a large numbers of which are the hallmark of periodontal inflammation.

3. Chairside, 1 minute result4. Good for patient motivation 5. Cheap6. Can be used for antibiotic

verification( efficacy/ compliance)

Disadvantages:1. Initial cost2. Unfamiliar technology3. No antibiotic specificity4. Inability to differentiate among the various

species of trponema5. Inability to identify the main putative

periodontal pathogens like A. actinomycetemconitans, P. gingivalis as they are non motile.

IMMUNODIAGNOSTIC METHODS Immunological assays employs antibodies that

recognize specific bacterial antigens to detect target micro-organisms.

A variety of procedures can be used such as:a) Direct and indirect immunofluorescent

assaysb) Flow cytometryc) Enzyme-linked immunosorbent assayd) Latex agglutination

IMMUNOFLUORESENCEPrinciple:Immunofluoresence is an antigen-antibody

reaction where antibodies are labelled with a fluorescent dye and antigen-antibody complex is visualized using ultra-violet microscope.

Immunofluorescent assays has been used mainly to detect A.a., P.g. in subgingival dental plaque.

Direct immunofluorescence: It employs both monoclonal & polyclonal

antibodies conjugated to a fluorescein marker that binds with the bacterial antigen to form a fluorescent immune complex detectable under a light microscope.

Indirect immunofluoresence: It employs a secondary fluorescein-

conjugated antibody that reacts with the primary antigen-antibody complex.

Identifies A.a & P.g. inn subgingival plaque samples.

FLOW CYTOMETRY/ CYTOFLUOROGRAPHY: It uses the principles of light scattering, light

excitation and emission of fluorochrome molecules to generate specific multi-parameter data from particles and cells in the size range of 0.5 µm to 40 µm diameter.

In this technique, plaque sample is taken from the site and bacterial cells are labelled with species-specific antibody and second fluorescein conjugated antibody.

This suspension is then introduced in a flow cytometer which seperates the bacterial cells into an almost single suspension by means of laminar flow.

It helps in detection and quantification of P.gingivalis in bacterial mixtures.

It is an expensive method.

ENZYME-LINKED IMMUNOSORBENT ASSAY: It is the quantitative immunological

procedures in which Ag-Ab reaction is monitored by enzyme measurements.

The term was given by Engvall & Perlma in 1971.

This was the first screening test commonly employed for HIV.

Useed primarily to detect serum antibodies to periodontal pathogens can also be used to quantify specific pathogens in subgingival samples using specific monoclonal antibodies.

EVALUSITE: A membrane immunoassay adapted for chair-

side clinical diagnostic use. It involves linkage between the antigen & a

membrane- bound revealed through a colorimetric reaction.

Designed to detect A.a., P.g., P.i.

LATEX AGGLUTINATION: Simple method based on binding of protein to

latex. Latex beads are coated with species-specific

antibody & when these beads come in contact with the microbial cell surface antigens, cross-linking occurs; clumping is visible in 2-5 minutes.

Advantages:1. Rapid2. Simple3. Great potential for chair side detection of

periodontal pathogens4. Higher senstivity & specificity for A.a., P.g. &

T.f.5. Does not requiring stringent sampling and

transport methodology to ensure bacterial viability.

Disadvantages:Not clinically available

ENZYMATIC METHODS: BANA This type of test depends upon the presence

of a virulence factor or other more general markers for microbial activity.

The pathologic nature of the subgingival microflora is inferred by the presence of an enzyme or other virulence factors.

Studies have focused on the presence of a trypsin-like enzyme in subgingival plaque that can hydrolyze the synthetic substance benzoyl-DL-argininenaphthylamide(BANA)

When hydrolysis takes place, it releases the chromophore β-naphthylamide, which turns orange red when a drop of fast garnet is added to the solution.

Diagnostic kits have been developed using this reaction(Perioscan).

Beck used this test as a risk indicator for periodontal attachment loss.

SUMMARIZATION OF BANA TESTa) Red group testingb) Chairside, 5 minute resultsc) A highly sensitive and specific enzymatic

assay for P.g., T.f. & T.d, the three most pathogenic periodontal microorganisms.

d) The use of the BANA test in clinical periodontal practice has proved to be of high value in aiding the diagnosis of periodontal disease and is of practical applicability in the bacteriologic monitoring of periodontally involved patients.

e) The test can be compared in card form(Perioscan) and in liquid form. The results are equivalent with the advantage that the perioscan method is easier to use and the results can be obtained during the sane visit when the laque sample is collected.

f) It only detects a very limited number of pathogens, its negative results do not rule out the presence of other important periodontal pathogens. Also, it may be positive at clinically healthy sites.

DIAGNOSTIC ASSAYS BASED ON MOLECULAR BIOLOGICAL TECHNIQUES NUCLEIC ACID PROBING: A probe is known as nuceic acid molecule

from a specific micro-organism artificially synthesized and labeled for its detection when placed with a plaque sample.

DNA probes use segments of single stranded DNA nucleic acids, labeled with an enzyme or radio-isotope that is able to hybridize to the complimentary nucleic acid sequence and thus detect the presence of target organism.

Hybridization refers to the pairing of complementary DNA strands to produce a double stranded nucleic acid.

Currently, most of the probes used are oligonucleotides ranging from 20-30 nucleotides.

It is used for the detection of A.a., P.g., P.i., F.n., T.d.

CHECKERBOARD DNA-DNA HYBRIDIZATION TECHNIQUE:

Sockransky et al developed this technique for the presence of upto 43 bacterial species.

The assay uses whole genomic, digoxigenin-labeled DNA probes and enables rapid processing of numerous plaque samples and is often used for research purpose.

POLYMERASE CHAIN REACTION: Uses a DNA replicating enzyme to amplify

target sequences of DNA. Standard PCR is not a quantitative

assessment of identified bacteria, although a technique called real time PCR does enable quantification.

Problems with DNA/ oligonucleotide probes:1. Need for validation of specificity of probe for

identification of similar species in a genus2. Lack of readily available information

concerning antibiotic sensitivty of the identified organism.

3. They require expensive and sophisticated technology.

Periodontist must therefore choose one of these two methods according to specific clinical objective:

To obtain rapid, specific detection with weak initial concentrations but for targeted periopathogens or to be non-specific and analyze the pathological activity with an antibiogram.

Hence, on the whole, nucleic acid techniques should replace cultivation methods as gold standard in microbiological diagnosisof progressive periodontitis.

Micro-iDEnt Plus DNA Periodontal Diagnostic Test Kit

This test kit identifies 11 major and minor bacterial pathogens found in periodontal infections; the pathogens are classified into four groups to simplify therapy and antibiotic selection.

The simple-to-use test involves inserting a paper point into the pocket or sulcus and sending to lab for testing.

Test results are provided electronically with downloadable documents with full color graphs for patient education and archiving in patient files.

Use of test helps identify at-risk patients, determine appropriate therapy, monitor compliance and create optimum recall schedule.

Application of diagnostic tests: Diagnostic tests will find application with

patients who have never received periodontal therapy. Baseline data for one or more tests may be part of the information collected when the patient is first examined.

Following the initial phase of periodontal therapy, there is often a question about the need for additional treatment, and a test that identifies the future, near-term risk for active disease may well be helpful in this situation.

While the reduction of tissue inflammation and excessive probing depth are fundamental clinical principles in periodontics, diagnostic tests may be used when patients are poor risks for periodontal surgery due to an existing medical condition or age.

Diagnostic tests are likely to be used for assessment of patients who have received comprehensive periodontal therapy and are on a maintenance schedule.

Identification of therapeutic endpoint that can be used to determine when adequate therapy has been provided.

The tests can be used to help establish the appropriate re-call interval (i.e., here the test is used to establish a therapeutic endpoint). This type of test would ideally have an in situ or inoffice format that could provide results before the next appointment is scheduled. The re-call interval could remain the same, could change, or more intensive therapy could be planned.

Last, a test may be utilized for patients considered refractory to conventional therapy. Since these tests analyze some aspect of the pathologic process (i.e., the presence of a putative pathogen), utilization may help identify why the patient is entering an active phase.

Consecutively, follow-up testing may provide a measure of the effectiveness of therapy.

CONCLUSION Although there are many potential biomarkers

for periodontal disease activity and progression numerous features still hamper the ability to use them as diagnostic tests of proven utility. There is still lack of a proven gold standard of disease progression. After all these years of intensive research, we still lack a proven diagnostic test that has demonstrated high predictive value for disease progression, has a proven impact on disease incidence and prevalence, and is simple, safe and cost effective.