Introduction

Collection and transport of specimen

Limitations of conventional periodontal diagnosis

Microbiological analysis

Bacterial culturing

Microscopic methods

Immunodiagnostic methods

Enzymatic methods

Molecular biology techniques

Conclusion

References

Diagnostic microbiology- involves the study of specimens

taken from patients suspected of having infection

Steps of a diagnostic cycle:

1) clinical request and provision of clinical information

2) collection and transport of specimen

3) laboratory analysis

4) Interpretation of the microbiology report and use of the

information

Request form:

Clinical condition of the patient

Age

Date of onset of illness

Recent/current antibiotic therapy

Antibiotic allergies

History of previous specimen

Specimens should be collected before antibiotic therapy as

it will alter the quality and quantity of the organisms

Exceptions-patients who are seriously ill, immunologically

compromised or not responding to a specific antibiotics

Specimens should be collected following appropriate

protocol

Specimens should be as fresh as possible- anaerobes,viruses

die..staphylococci, coliforms may multiply…at room

temperature

Transport specimens in an appropriate medium and as fast

as possible

Medium should be compatible with the organisms

Transport specimen in safe, robust containers to avoid

contamination

For assessing organisms in periodontal infections, an

appropriate subgingival plaque sample has to be obtained

Obtaining it from appropriate sites and in adequate quantity

Mombelli et al- four individual subgingival specimens from

each quadrant

Sample collected and transported in an anaerobic

environment with minimal transport time

Curettes vs paper points

Conventional diagnosis of gingivitis depends on clinical

signs and symptoms

Conventional diagnosis of periodontitis depends on clinical

attachment loss and radiographic bone loss

Tells us about the extent and severity of disease

Does not give information about the cause of the disease,

on the patients susceptibility to disease, whether the disease

is progressing, whether it is in remission, or whether the

response to therapy will be positive or negative.

Disease process is mulitfactorial in origin in which

periodontal pathogens, host response , and genetic,

systemic, and behavioural risk factors interplay to develop

the disease process

Many publications have reported that certain micro-

organisms from the subgingival microbiota , particularly

gram negative anaerobes, are the major etiologic factors of

chronic and aggressive periodontitis

More than 300 species isolated from different individuals

40 species from a single site

According to criteria described by Socransky-

1) Strong evidence for Aa, Pg, Tf

2) Moderate evidence for campylobacter rectus,

eubacterium nodatum, fusobacterium nucleatum,

peptostreptococcus micros, prevotella intermedia,

prevotella nigrescens, streptococcus intermedius, and

spirochetes such as treponema denticola

Microbiological tests are useful..

1) To identify putative pathogens and supporting the diagnosis of various forms of periodontal disease

2) To serve as indicators of disease initiation and progression and healing

3) To determine which periodontal sites are at higher risk for active destruction

4) To moniter periodontal therapy

5) To aid in treatment planning of patients with aggressive or non responding periodontitis by helping the doctor in selection of adjunctive antimicrobial therapy

Microbiologic testing for the presence or absence of Pg and Aa

to distinguish subjects with aggressive periodontitis from

those with chronic periodontitis has its limitations-according

to systematic review by Mombelli et al.

Bacterial culturing

Still considered gold standard when determining the

performance of new microbial diagnostic methods

Originally used by Louis Pasteur. He used urine and

meat broth. First solid medium...cooked cut potato used

by Robert Koch

Koch used gelatin to solidify liquid media but not

successful as gelatin liquified at 24˚c and also by many

proteolytic bacteria

Frau Hesse suggested use of agar to solidify liquid media

Advantage of this method

1) Clinician can obtain relative and absolute counts of the

cultured species

2) Only in vitro method able to assess for antibiotic

susceptibility of the microbes

Disadvantage

1) Can only grow live bacteria. Thus strict sampling and

transport conditions are essential

2) Some putative organisms such as treponemas species

and Tf are fastidious and difficult to culture

3) Low sensitivity. Detection limits average 103 to 104

bacteria. Thus low number of specific pathogens go

undetected.

4) Requires sophisticated equipments and experienced

personnel

5) Time consuming and expensive

Solid or liquid media used for bacterial or fungal growth

Cultured cells derived from animals and humans used

for viral growth

SolidSemi-

solid

Liquid

Blood agar most widely used bacterial culture medium

Solid media..discrete visible growth…liquid

media..diffuse growth

Bacterial colonies

Solid media..

1) Distinct colonies

2) Pigmentation or hemolysis may be present.

Identification made easy

3) Quantification of the organisms as colony forming

units (CFU)

Agar now universally used for preparing solid media

Agar-agar

Obtained from sea weeds

Chief constituent a long chain polysaccharide

Also contains inorganic salts and small quantities of a

protein like substance

No nutritive value , not affected by growth of bacteria

Hydrolysed at high temperatures at high acid or alkaline

ph

Melts at 98˚c and sets at 42˚c

2% agar employed for solid media

Manufactured in long shreds or powder form

Advantages..

1) For obtaining bacterial growth from blood or water when

large volumes have to be tested

2) For preparing bulk cultures for preparation of antigens or

vaccines.

3) To test biochemical activities of bacteria for identification

purposes

4) To promote growth of small number of bacteria in

specimens contaminated with antibiotics. fluid medium

dilutes antibiotics

Disadvantage of liquid media

1) Difficult to isolate different types of bacteria from

mixed populations

2) Bacteria growing in liquid media may not exhibit

characteristics for their identification

Primary antibiotic tests /secondary antibiotic tests

Simple media- eg nutrient broth

Complex media

Synthetic or defined media -simple peptone water

medium,1%peptone with 0.5%NaCl

Enriched media -blood agar, chocolate agar, egg media

Enrichment media- liquid in nature. Eg tetrathionate

broth.

Selective media -solid media. Similar to enrichment

media but solid in nature. Eg desoxycholate citrate

medium

Indicator media- sulphite in Wilson and Blair

medium, potassium tellurite in McLeods medium

Differential media -eg MacConkeys medium.

Lactose fermenters as pink colonies and non lactose

fermenters as colourless colonies

Sugar media -Any medium with fermentable substance

Transport media- Stuarts medium for gonococci

Thyoglycolic acid as a reducing agent.

Viral transport medium -solutions containing proteins

and balanced salts which stabilizes the virus during

transport and antimicrobials to kill bacterias

Anaerobic media- Robertsons cooked meat medium

Introduction

Collection and transport of specimen

Limitations of conventional periodontal diagnosis

Microbiological analysis

Bacterial culturing

Microscopic methods

Immunodiagnostic methods

Enzymatic methods

Molecular biology techniques

Conclusion

References

Bright field or standard microscopy

Routinely used

Stained smears examined with oil emersion objective(x100)

using the x10 eye piece obtaining a magnification of x1000

Wet films are examined with a dry objective(x40)…used to

demonstrate motility of bacteria

Dark ground microscopy

Specimen is illuminated obliquely by a special

condenser so that light rays do not enter objective

directly

Instead the organisms appear bright, as the light rays hit

them, against the dark background

Helpful in studying motile organisms

But most putative periodontal pathogens are non

motile(Aa, Pg, Tf)

Phase contrast microscopy

Rarely employed in diagnostic microbiology

May be used to define the detailed structure of unstained

microbes

Fluorescence microscopy

This method employs the principle of emission of a

different wavelength of light when light of one

wavelength strikes a fluorescent object

UV light normally used

Bacteria and cells stained with fluorescent dyes such as

auramine.

Used widely in immunology – antibodies tagged with

fluorescent dyes to detect bacterial antigens

Electron microscopy

Light waves replaced by a beam of electrons which

allows resolution of extremely small organisms such as

viruses

Can be used in diagnostic virology

Direct examination of specimens can be done but

approximately one million virus particles are needed for

such visualization.( eg rotavirus, hepatitis A virus)

Immunoelectron microscopy

Immunoelectron microscopy - Clumps of viral particles

can be obtained by reacting the sample with antiviral

antibody .Useful for some viruses such as hepatitis A

virus

Immunoferritin test -Ferritin can be conjugated with

antibody,and such a labelled antibody reacting with an

antigen can be visualised under electron microscope

Immunoenzyme test -Enzymes such peroxidase, glucose

oxidase, phosphatases and tyrosinase can be conjugated

with antibodies and such a labelled antibody reacting

with an antigen can be seen under electron microscope

Gram stain technique

Dry film heat fixed by gently passing through a flame

Then flood with crystal violet for 15 seconds and wash

excess

Flood with Lugol’s iodine for 30 seconds to fix stain and

wash excess

Decolorize with acetone or alcohol for about 5 sec

Wash with water

Counterstain with dilute carbolfuchsin for 30 seconds

Wash with water and blot dry

Gram positive bacteria- They retain the violet stain by

resisting decolourization and are stained blue

Gram negative bacteria- They loose violet stain during

decolourization and are therefore counterstained with

pink

Ziehl –Neelsen technique

Organisms are exposed to hot concentrated carbolfuschin for

5 min, decolourized with acid and finally counterstained with

methylene blue or malachite green

Bacilli will stain red against a blue background

Useful for tubercle bacilli, which are difficult to stain with

gram stain due to thick cell wall

An antigen has been defined as any substance which, when

introduced parenterally into the body, stimulates the

production of an antibody with which it reacts specifically and

in an observable manner

Immunoglobulins- Proteins of animal origin endowed with

known antibody activity and for certain other proteins related

to them by chemical structure.The definition includes besides

antibody globulins, the abnormal proteins found in myeloma,

macroglobulinemia, cryoglobulinemia and naturally occuring

subunits of immunoglobulins

Synthesised in plasma cells and lymphocytes

All antibodies are immunoglobulins but all

immunoglobulins may not be antibodies

IgA, IgG, IgM, IgD, IgE

Advantage-stringent sampling and transport

methodology not required

Higher specificity and sensitivity than bacterial culturing

Disadvantage-Limited to the number of antibodies tested

Antibiotic sensitivity cannot be studied

Lack of validity of well controlled clinical studies

Use of monoclonal antibodies for high specificity

Immunofluorescence

Fluorescence is the property of absorbing light rays of one

particular wavelength and emitting rays with a different

wavelength

Fluorescent dyes show up brightly under ultraviolet light

as they convert ultraviolet into visible light

Coons and his colleagues (1942)showed that fluorescent

dyes can be conjugated to antibodies and such labelled

antibodies can be used to locate or identify antigens in

tissues

Direct immunofluorescence test

Can be used for identification of bacteria, viruses or

other antigens

Antibodies specific for the particular antigen are labelled

with a fluorescent dye

Eg- Used for diagnosing rabies by detection of rabies

virus antigen in brain smears

Pathogens can be identified and quantified by this

method by visualizing under a microscope

Disadvantage- Separate fluorescent conjugates have to

be prepared against each antigen to be tested

This disadvantage is overcome by the indirect

immunofluorescence test

Indirect immunofluorescence test

In this method a secondary fluorescein-conjugated

antibody is used that reacts with the primary antigen-

antibody complex

This method also useful for identification and

quantification of the pathogen

Eg- Fluorescent treponemal antibody test for the diagnosis of syphilis

A drop of test serum is placed on a smear of T pallidum on a slide

After incubation slide washed to remove all free serum leaving behind only the antibody globulin, if present , coated on the surface of the treponemas

Smear then treated with fluorescent labelled anitserum to human gammaglobulin

The fluorescent conjugate reacts with with antibody globulin bound to treponemas

Treponemas seen as bright objects against dark background under ultraviolet illumination

VDRL-Non treponemal test in which cardiolipin,

lecithin and cholesterol mixture is used as antigen

Clumping of cardiolipin occurs in presence of antibodies

to treponema pallidum

Not a confirmatory test

Both tests used as T pallidum does not grow in

laboratory media

A single antihuman globulin fluorescent conjugate can be employed for detecting human antibody to any antigen

Fluorescent dyes may also be conjugated with complement

Complements can be employed for the detection of antigen or antibody

Antigens also take up fluorescent labelling but not as well as antibodies do

for detection of antibodies by immunofluorescence the “sandwich” technique can be used

Commenly used fluorescent dyes- fluorescein isothiocynateand lissamine rhodamine exhibiting blue-green and orange-red fluorescence respectively

Immunofluorescent assay has been mainly used to detect

Aa and Pg

IFA comparable to bacterial cultures in its ability to

identify periodontal pathogens in subgingival dental

plaque samples- Zambon et al

IFA microscopy does not require viable bacterial cells

Sensitivity of these assays range from 82% to 100% for

detection of Aa and from 91% to 100% for detection of

Pg

Specificity values are 88% to 92% and 87% to 89%

respectively

Flow cytometry /cytofluorography

Used for rapid identification of oral bacteria

Involves labelling of bacterial cells from patient plaque samples with both species specific antibodies and a second fluorescein conjugated antibody

The suspension is then introduced into the flow cytometer, which separates the bacterial cells into an almost single cell suspension by means of a laminar flow through a narrow tube

Highly sophisticated instrument

Very costly

Radioimmunoassay

Radio isotopes used as labels

Binder-ligand assay

Substance whose concentration is to be determined is

termed as analyte or ligand

The binding protein which binds to the ligand is called

the binder

First described by Berson and Yallow in 1959 for which

they were awarded Nobel Prize in 1977

Permits the measurement of analytes upto

picogram

Can be used for quantitation of hormones, drugs,

tumour markers, viral antigens and IgE

It is a competitive binding assay in which fixed

amounts of antibody and radiolabelled antigen

react in the presence of unlabelled antigen

The concentration of the test antigen can be

calculated from the ratio of bound and total

antigen labels, using a standard dose response

curve

Enzyme immunoassay

Enzymes used as labels

First introduced in 1966

Very versatile, sensitive, simple and economic procedure

Absence of radiation hazard

Newer automatic machines have been introduced

Test kits available

Two basic types 1)homogeneous 2)heterogeneous

Homogeneous EIA

No need to separate the bound and free fractions

The test can be completed in one step with all the

reagents added simultaneously

Used only for haptens such as drugs

Eg-Enzyme multiplied immunoassay technique (EMIT)

to detect drug molecules like opiates, cocaine,

barbiturates, amphetamine in serum

Heterogeneous EIA

Requires separation of free and bound fractions either by

centrifugation or by absorption on solid surfaces and

washing

Multistep procedure

Reagents added sequentially

Eg- enzyme linked immunosorbent assay (ELISA)

ELISA

Involves use of an immunosorbent

An absorbing material specific to the antigen or antibody

Hence the name enzyme linked immunosorbent assay

Eg-1) Particulate-cellulose, agarose

2) Solid phase- polystyrene, polyvinyl, polycarbonate

tubes or microwells

3) Membranes or discs -polyacrylamide, paper, plastic

Done using 96-well microtitre plates suitable for automation

Always use positive and negative controls

Examples described above are of simple non competitive

sandwich ELISA

Competitive ELISA - The test can be made more specific by

making serum antibody and enzyme labelled antibody

compete for the binding sites on the antigen

Immunoglobulin specific elisa -eg IgM specific ELISA

The intensity of colour produced depends on the

concentration of the antigen or antibody being detected and

is usually read photometrically for optimal quantification

Simple modification of ELISA

Each specimen tested in a separate disposable casette

Test is rapid taking only 10 minutes compared to 2-4 hrs

taken by microplate ELISA

No need for microplate washers or readers

Result read visually

Inbuilt positive and negative controlls

Used for detection of HIV type 1 and 2 antibodies

Also known as membrane immunoassay

Useful for chairside clinical diagnosis

Eg- Evalusite. Designed to detect Aa and Pg with105 and

106 detection limit respectively

Method

Specific type 1 and 2 antigens are immobilised at separate fixed sites on nitrocellulose membrane in cassette

Test serum added on membrane and allowed to filter into absorbent material placed below it in the cassette base

Antibody if present will bind to the appropriate antigen

After washing, enzyme labelled antihuman immunoglobulin antibody is added

After further washing, a substrate yielding a colouredproduct is added

positive result indicated by a coloured spot

Immunoelectroblot techniques(western blot test)

They combine the sensitivity of enzyme immunoassay, with

much greater specificity

Used for identification of proteins

Three steps

1) Separation of ligand-antigen component by

polyacrylamide gel electrophoresis

2)Blotting of electrophorised ligand fraction on nitrocellulose

membrane strips

3)Identification by radio labelled or enzyme labelled

antibodies as probes

Confirmatory test for the diagnosis of HIV antibodies in

sera

A similar technique for analysis of DNA is known as

southern blot technique and for analysis of RNA is known

as northern blot technique

Agglutination

Slide agglutination

Latex agglutination

Latex beads coated with species-specific antibodies

Agglutination when contact with antigen in 2-5 minutes

Tannerella forsythia (Tf), Porphyromonas gingivalis (Pg),

Treponema denticola (Td), and Capnocytophagia species

share a common enzymatic profile

Trypsinlike enzyme

The activity of this enzyme can be measured wiyh the

hydrolysis of the colourless substrate N-benzoyl-d L-

arginine-2-naphthylamide(BANA)

Release of chromophore ß-naphthylamide on its

hydrolysis, which turns orange red when a drop of fast

garnet is added to the solution

Perioscan

Loesche proposed the use of this BANA reaction in subgingival plaque samples to detect any of the above mentioned periodontal pathogens and thus serve as a marker of disease activity

Loesche showed that shallow pockets exhibited only 10% positive BANA reactions, whereas deep pockets (7mm) exhibited 80 % to 90% positive BANA reactions

Beck used the BANA test as a risk indicator for periodontal attachment loss

Positive results indicate that one or more of Tf, Pg, Td bacteria are present at the sampled sites

Perioscan

Potential difficulties- May be positive at clinically

healthy sites

Not proven whether this test can detect sites undergoing

periodontal destruction

It only detects a very limited number of periodontal

pathogens and hence its negative result does not rule out

the presence of other important periodontal pathogens

The principles of molecular biology techniques reside in

the analysis of DNA, RNA and the structure or function of

protein

Genetic material of a bacterium is composed of a

chromosomal DNA and transferring RNA (tRNA),

ribosomal RNA (rRNA), and messanger RNA (mRNA)

Chromosomal DNA is dispersed in the bacterial cell

without a membrane envelope

Diagnostic assays employing molecular biology

techniques require specific DNA fragments that recognize

complementary-specific bacterial DNA sequences from

target microorganisms

Thus such techniques require the ability to extract

bacterial DNA from the plaque samples and amplify the

specific DNA sequence of the target periodontal

pathogens

Nucleic acid probes

Probe -A known nucleic acid molecule (DNA or RNA) from

a specific microorganism artifically synthesized and labeled

for its detection when placed with a plaque sample

These probes use segments of a single stranded nucleic acid,

labelled with an enzyme or radioisotope that is able to

“hybridize” to the complementary nucleic acid sequence and

thus detect the presence of target microorganisms

Hybridization - Refers to the pairing of complementary DNA

strands to produce a double-stranded nucleic acid. The

nucleotide base pair relationship is so specific that the strands

cannot anneal unless the respective nucleotide strand

sequences are complementary

Nucleic acid probe

All hybridization methods use radiolabeled or

fluorescence labeled DNA probes that bind to the target

DNA of interest, thus allowing visualization

DNA probes may target whole genomic DNA or

individual genes

Whole genomic DNA is more likely to cross react with

non target microorganisms because of the presence of

homologous sequences between different bacterial

species

Currently most of the probes used are oligonucleotides,

ranging from 20 to 30 nucleotides

Whole genomic probes for detection of Aa, Pg, Td have

been developed and tested- eg DMDx, Omnigene

Sensitivity of 96% and specificity of 86% for Aa

and sensitivity of 60% and specificity of 82% for Pg in

pure laboratory isolates when compared to culture

according to Van Steenberghe et al

Sensitivity and specificity reduced significantly in

clinical specimen, suggesting cross reactivity with

unknown bacteria in subgingival plaque samples

Oligonucleotide probes complementary to variable

regions of the 16S rRNA bacterial gene

16S rRNA genes contains both- 1)Regions shared by

different bacteria and 2) Short stretches of variable

regions shared only by specific organisms of the same

species or genus

When compared to cultures in clinical samples,

effectiveness of 100% in detecting Aa and Pi and 91% in

detecting Pg with oligonucleotide probes-Savitt et al

In detecting pathogens in samples from periodontitis

patients : IFA>DNA probes>cultures

Developed by Socransky et al for the detection and

estimation of levels of 40 bacterial species often found in

the oral cavity

Whole genomic, digoxigenin-labeled DNA probes used

Facilitates rapid processing of large number of plaque

samples with multiple hybridization for upto 40 oral

species in a single test

DNA probes used in this technology are adjusted to permit

detection of 104 cells of each species

Sophisticated laboratory equipment

Expertise

Thus assay not generalized for diagnostic purposes

Applicable for epidemiologic research and ecologic studies as it does not require viable bacteria and allows the assessment of large number of plaque samples and multiple species

This method resulted in higher prevalence figures for half the species tested (Pg, Pi, Tf and Fn) and statistically significant higher bacterial counts for the majority of species when compared to cultures for identification of subgingival bacteria-Papapanou et al

Developed in 1985

Has emerged as the most powerful tool for the amplification of

genes and their RNA transcripts

Used almost universally to study DNA and RNA obtained

from a variety of tissue sourses

PCR allows large quantities of DNA to be obtained in a

simplified and automated manner

Steps :

Isolation of DNA from a fresh tissue specimen

Heating of the complementary double strands to split

DNA into single stranded forms which are intended to

act as template dictating the nucleotide sequence in vitro

Amplification using a DNA polymerase that requires a

primer

A primer is a known short oligonucleotide sequence

corresponding to the border of the region that is

amplified

For obtaining amplified segments of constant length

and in large quantities, a second primer, complementary

to the opposed chain, must be used to bind the template

and flank the region of interest

This amplification can be performed several times

known as cycles

In each cycle, complementary chain denaturation,

primer hybridization, and primer extension by means of

the polymerase takes place

With each cycle there is an exponential increase in the

quantity of DNA

Temperature is critical throughout the cycle to control

the double chain denaturation and the stability of the

hybridization between the model fragment and the

primer

In 1988, thermostable DNA polymerase isolated from

organism Thermus aquaticus known as Taq-polymerase

was developed

This Taq-polymerase has allowed automatization of the

reaction using specific appliances called thermocyclers

This sequenced DNA is then detected and visualized

through electrophoresis in agarose gel and ethidium

bromure, obtaining a qualitative signal

Ashimoto et al developed a 16S rRNA based PCR

detection method to determine the prevalence of Aa, Tf,

Pg, Pi, Td.

Compared to culture where detection limit is 104-105

cells, PCR can detect cells as low as 25-100

Microbial tests using standard PCR are extremely

sensitive and specific

But they provide only qualitative information

Therefore their use for diagnostic and prognostic

purposes in clinical use is limited

Quantitative PCR methods

1) Endpoint PCR

2) Real time PCR

This method demonstrated high degree of sensitivity,

specificity, and reproducable quantification

Requires expensive laboratory equipment

Thus limited use in routine diagnostic clinical

microbiology

Textbook of Microbiology,5th edition-R.Ananthanarayan

Essential Microbiology for Dentistry- L.P.Samaranayake

Short Textbook of Medical Microbiology- Satish Gupte

Carranza’s Clinical Periodontology,10th edition

Molecular Microbial Diagnosis-Bruce Paster, Floyd Dewhirst

periodontology 2000,vol 51,2009.38-41

Microbiological diagnostic testing in the treatment of periodontal

disease-Peter Loomer periodontology 2000,vol 34,2004,49-53

Microbial testing in periodontics:value, limitations and future

directions-Luciana Shaddox, Clay Walker periodontology 2000,vol

50, 2009, 25-38

Contemporary Oral Microbiology and Immunology- Slots,

Taubman.