CARIES ACTIVITY TESTS

CONTENTS

Introduction

Purpose of caries activity tests

Ideal requisites of caries activity tests

Caries activity tests in dental office

Some new caries activity tests

Cariogram

Limitations of caries activity tests

References

INTRODUCTION

Dental caries (latin word carius=rottenness) It is defined as a progressive, irreversible microbial disease of multifactorial nature

affecting the calcified tissues of the teeth, characterized by demineralization of the inorganic portion and destruction of the organic portion of the tooth.

It is a carbohydrate-modified transmissible local infection with saliva as a critical regulator.

Caries activity tests measure the degrees to which the local environment challenge (eg: dietary effect on microbial growth and metabolism) favours the probability of carious lesions

PURPOSE

For the clinician:

To determine the need for caries control measures.

As an indicator of patient co-operation.

To act as an aid in timing of recall appointments.

As a guide to insertion of expensive restorations.

To aid in determination of prognosis.

As a precautionary signal to orthodontist in placing bands.

For the research worker:

As an aid in selection of patients for caries study.

To help in screening of potential therapeutic agents.

To serve as an indicator of periods of exacerbation and remission.

IDEAL REQUISITES

According to Snyder

1. Have a sound theoretical basis. 2. Show maximum correlation with clinical status 3. Should have validity, reliability and feasibility 4. Be accurate w.r.t duplication of results 5. Be simple. 6. Be inexpensive 7. Take little time

Caries activity tests for the dental office:

Dental caries is multifactorial and of complex etiology. No test is present which can fully explain or predict the disease.

With caries activity tests information can be obtained on selected factors of importance for the process.

The tests should be simple and inexpensive and rapid and accurately reflect the 3 overlapping circles presented by KEYS(1962).

In the light of these requirements and circles, caries activity tests can be grouped under:

1. Bacterial challenge- determination of mutans streptococci as an indicator of relative risk.

2. Diet- determination of lactobacilli as an indicator of sugar content in the diet.

3. Remineralization potential- salivary flow rate and buffer capacity as an indicator of potential biologic repair.

4. Host susceptibility- caries experience as an indicator of past activity.

When should the tests be used?

Clarify the reasons behind an on going disease and formulate and motivate the preventive strategy to the patient.

Determine the effect of a causal treatment at follow up visits. Predict caries development (ie; make a prognosis) at check up visits.

CARIES ACTIVITY TESTS

Test to identify Streptococcus mutans Plaque/toothpick method Saliva/tongue blade method S mutans adherence method S mutans dip-slide method S mutans replicate technique

Tests for Lactobacilli Lactobacilli count testDip slide method

Tests to check acidogenicity Snyder testAlbans test

CARIES SUSCEPTIBILITY TESTS

Reductase test Buffer capacity test

Fosdick calcium dissolution test Dewar test

NEW CARIES ACTIVITY TESTS

Cariostat Caries risk test

Oratest

Plaque/toothpick-method

Action- involves simple screening of a diluted plaque sample streaked on selective culture medium.

Equipment-1.sterile toothpicks

2.sterile ringer’s solution(5ml)

3.platinum loop

4.mitis salivaris agar plates

containing sulphadiametine(1g/l)

5.incubator

Plaque samples are collected from gingival 3rd of buccal tooth surfaces(one from each quadrant) and placed in ringer’s solution

The sample is shaken until homogenized

Plaque suspension is streaked across a mitis salivarius agar plate

After aerobic incubation at 370 C for 72 hrs, cultures are examined under a low power microscope and the total colonies in ten fields are recorded

Results of a s mutans screening test

Grade Colonies/10fields No. of cases No. of cases with new lesions

1 None 21 4

2 <8 11 5

3 >8 12 12

Saliva/tongue blade method

Action: estimates the no. of s mutans in mixed paraffin stimulated saliva when cultured on mitis salivarius bacitracin(MSB) agar.

Equipment: 1.paraffin wax

2.sterile tongue blades

3.disposable contact petridish

containing MSB agar

4.incubator

Subjects chew paraffin wax for 1 min.

Sterile tongue blades given -subjects rotate in their mouths 10 times, so that both the sides of the tongue blade are thoroughly inoculated by the subject’s flora.

Tongue blade is withdrawn through closed lips. Both the sides of the tongue blade are then pressed onto an MSB agar plate which is then incubated at 370 c for 48 hrs.

Counts of more than 100CFU by this method are proportional to greater than 106 CFU of S mutans per ml of saliva by conventional methods.

For field studies the plates are placed into plastic bags containing expired air, which are then sealed and incubated.

An updated or simpler version of this method, strip mutans (orion diagnostica), uses a liquid medium with a long shelf life, to which the bacitracin is added just before incubation.

A plastic spatula instead of wooden blade is used. Colonies of mutans streptococci grow directly on the spatula, which is then removed from the liquid medium, drained and placed under microscope for counting.

Advantages:

1. Simple and practical method for field studies

2. Avoids the necessity of collecting saliva

3. Requires no transport media/dilution stops

S mutans adherence test

Action: categorizes salivary samples based on the ability of s mutans to adhere to glass surfaces when grown in

sucrose containing broth.

Procedure:

Unstimulated saliva (0.1ml) is inoculated in MSB broth. The inoculated tubes are set at 600 angle and incubated aerobically at 370c for 24 hrs. After growth has been observed, supernatant medium is removed and cells adhering to the glass surface are examined macroscopically.

Scoring

- No growth expressed S mutans<10

4CFU/ml of saliva

+ Few deposits ranging from 1 -10 S mutans<10

4CFU/ml of saliva

++ Scattered deposits of smaller size

+++ Numerous minute deposits with more than 20 large deposits S mutans>10

5CFU/ml of saliva

S MUTANS DIP-SLIDE METHOD

Action: these tests comprise of Dentocult SM, Cariescreen SM.

These tests classify salivary samples according to estimates of s mutans colonies growing on modified mitis salivarius agar.

Equipment: paraffin wax, plastic slides coated with MS agar, CO2 tablets, buffered diluent.

Procedure A: (Dentocult SM)

Subject chews paraffin wax and stimulated saliva is collected for 5 mins

Saliva is poured over agar coated slide, totally wetting the surface, and excess is allowed to drain off.

Slides are dried for 10-15mins, bacitracin disks are placed in middle of the inoculated agar

A co2 tablet is inserted in the tube containing the slide, which is then incubated for 48hrs

A zone of inhibition 10-20 mm in diameter is formed around each bacitracin disk

S mutans appears as blue colonies growing within the zone of inhibition.

The colony density is compared with a model chart

Dentocult SM strip mutans test

Sample collection using Screening Strips Sample application using Site Strips

Incubation (48 hours) Interpretation (Screening Strips) Interpretation (Site Strips)

0 Negligible

1 <1,00,000 S.mutans CFU/ml

2 1,00,000-10,00,000 S.mutans CFU/ml

3 >10,00,000 S.mutans CFU/ml

Procedure B- (Cariescreen SM)

Bacitracin tablet is placed in buffered diluent and allowed to dissolve completely.

Subject chews paraffin wax for 15-20 sec swallowing the saliva.

While continuing to chew the wax the subject expectorates approx 1-2 ml of stimulated saliva directly into diluent vial, which is then capped and gently mixed by repeated inversion.

The dip slide coated on both sides with MSB agar( without bacitracin), is then immersed for few sec in the buffered diluent containing bacitracin and patient’s saliva.

A co2 tablet is placed in the empty dip slide vial, and 2 drops of water are added.

The dip slide is replaced in its vial and cap is securely tightened.

The vial is placed upright for 48hrs at 370 C in an incubator and then allowed to stand at room temp overnight.

The colony density on agar is compared with a reference colony density chart.

This test correlates strongly with actual laboratory methods of quantifying s mutans by standard culturing.

The s mutans colonies can be recognized when viewed through a magnifying lens by their opaque, highly convex appearance and irregular shape.

S mutans replicate technique

Action: localizes s mutans colonies on tooth surfaces using a solid impression matrix composed primarily of sucrose and commercial gum base.

Procedure: imprint of tooth surface to be sampled is obtained by pressing the matrix against it.

Matrix is washed for several sec in water to remove non adherent cells and saliva.

Matrices are placed in liquid broth and incubated at 370 C overnight and examined directly for overgrowth of s mutans colonies at specific sites.

Lactobacillus colony count test

First introduced by HADLEY in 1933.

Action: estimates the no. of acidogenic and aciduric bacteria in the patient’s saliva by counting the no. of colonies appearing.

Equipment: saliva collecting bottles, paraffin, 2 9ml tubes of saline, 2 agar plates, 2 bent rods, incubator, Quebec counter and pipettes.

Procedure:

Saliva is collected by having the subject chew paraffin before breakfast and then collecting the saliva in a bottle.

Specimen is shaken to mix it.

A 1:10 dilution is prepared by pipetting 1ml of saliva sample into a 9ml tube of sterile saline solution.

This is shaken and a 1:100 dilution is made by pipetting 1ml of 1:10 dilution into another 9ml tube of sterile salt solution.

0.4ml of each dilution is spread on the surface of an agar plate.

The plates are labelled and incubated at 370C for 3-4 days.

A count of the no. of colonies is then made by using quebec counter.

Results

No. of lactobacilli per ml saliva Caries activity

0-1000 Little or none

1000-5000 Slight

5000-10,000 Moderate

Advantages:

1. Useful for monitoring the effectiveness of restorative dentistry and care completion.2. Simple to carry out.3. Useful as a screening test for caries activity in large groups.

Disadvantages:

1. The results are not available for several days.

2. Cost is relatively high.

3. Inaccurate for predicting the onset of disease.

4. Does not completely exclude the growth of other relatively aciduric organisms.

5. Counting is a tedious procedure.

Dip slide method

Dentocult LB

In this method undiluted paraffin stimulated saliva is poured over a plastic slide that is coated with LBS agar.

Excess saliva is drained off and the slide is placed into the sterile tube, which is tightly closed and incubated at 350C-370C for 4 days.

The colony density on the slide is not counted instead is compared with a model chart and classified as about 1000, 10,000, 1,00,000 or 10,00,000 aciduric organisms/ml of saliva.

This method gives a highly significant correlation with the conventional lactobacilli count and a statistical significant correlation with the caries activity.

Sample collection Sample application

Incubation (4 days) Interpretation

Advantages:

1. Technique is simple, and results are easy to read.

2. Cost is reasonable.

Snyder test

Action: measures the rapidity of acid formation when a sample of stimulated saliva is inoculated into glucose agar with bromocresol green as color indicator.

The test is also a measure of acidogenic and aciduric bacteria.

Procedure:

Saliva is collected before breakfast by having the subject chew paraffin.

A tube of snyder glucose agar is melted and then cooled to 500C.

The saliva specimen is shaken vigourously for 3 mins. Then 0.2 ml of saliva is pipetted into the tube and incubated at 370C.

The color change of the indicator is observed after 24, 48, 72 hrs of incubation by comparison with an uninoculated tube against a white background.

Color changes in snyder test

24 hrs 48hrs 72 hrs

If yellow

Marked caries susceptibility

If yellow

Definite caries susceptibility

If yellow

Limited caries susceptibility

If green

Continue to incubate& observe at 48 hrs

If green

Continue to incubate & observe at 72 hrs

If green

Caries inactive

Incubation at 370C

Glucose agar

Medium with

bromocresol

Green indicator

+

0.2cc of saliva

Early color change to light green Extreme color change after 24 hours

Advantages-

1. Test is simple and requires simple equipment.

2. Test takes only 24-48 hrs

3. Cost is moderate.

4. Correlation with clinical trials

Disadvantage of snyder and lactobacillus count test:

Neither of the test can predict the extent of expectancy of caries with any reliability for one individual.

Several modifications of snyder test have been proposed to further simplify the method for use in dental office:

1. A smaller volume(0.2ml) of culture media is inoculated with saliva using calibrated wire loop. This avoids use of pipettes and saves medium and space.

2. Alban’s method uses less agar in the medium so that tubes do not require melting. There is no attempt to quantitate the salivary inoculum, which is drooled directly into the tube.

3. The buccal surfaces are swabbed and the cotton applicator incubated in semifluid snyder medium. This has advantage of culturing directly from plaque.

CARIES SUSCEPTIBILITY TESTS

Reductase test

Action: measures the rate at which an indicator molecule diazoresorcinol, changes from blue to red to colorless or leukoform on reduction by mixed salivary flora.

The test measures the activity of single enzyme, reductase (Rapp).

This enzyme is involved in some very definite and limiting reactions in the formation of products dangerous to tooth surface.

Procedure:

Saliva is collected by chewing a special flavored paraffin and expectorated directly into the collection tube.

When the saliva reaches the calibration mark(5ml) the reagent cap is replaced.

The sample is mixed with a fixed amount of diazoresorcinol, the reagent upon which reductase enzyme is to react.

The change in color after 30sec and after 15min is taken as measure of caries activity.

Test results vary with time after food intake and after brushing.

It has been proposed that this test is mainly a measure of oral hygiene status of the individual.

Advantages:

1. No incubation required

2. Quick results

Disadvantages:

1. Test results vary with time after food intake and after brushing.

Buffer capacity test

Action: measures the no. of millilitres of acid required to lower the ph of saliva through an arbitrary pH interval, such as from pH 7.0 to 6.0, or the amount of acid or base necessary to bring color indicators to their end point.

Equipment: pH meter and titration equipment, 0.05N lactic acid, 0.05N base, paraffin and sterile glass jars containing a small amount of oil.

Procedure:

10 millilitres of stimulated saliva are collected under oil atleast 1 hour before eating.

5ml of this are measured into a beaker. After correcting the pH meter to room temp. The pH of the saliva is adjusted to 7.0 by addition of lactic acid or base.

Lactic acid is then added to the sample until a pH of 6.0 is reached. The no. of the milliliters of lactic acid needed to reduce pH from 7.0 to 6.0 is a measure of buffer capacity.

There is an inverse relationship between buffering capacity of saliva and caries activity.

The saliva of the individuals whose mouths contain considerable amount of carious lesions frequently has a lower acid buffering capacity than the saliva of those who are relatively caries free.

This test however does not correlate with caries activity.

Fosdick calcium dissolution test

Action: measures the milligrams of powdered enamel dissolved in 4hrs by acid formed when the patient’s saliva is mixed with glucose and powdered enamel.

Equipment: powdered human enamel, saliva collection bottles, sterile test tubes, test tube agitation equipment, and equipment for determining the calcium content of saliva.

Procedure:

Twenty five milliliters of gum stimulated saliva collected.

Part of this is analyzed for calcium content.

The rest is placed in an 8 inch sterile test tube with about 0.1g of powdered human enamel.

The tube is sealed and shaken for 4hrs at body temp after which it is again analyzed for calcium content.

The chewing of gum to stimulate the saliva produces sugar, if paraffin is used a concentration of about 5% glucose is added.

Advantages:

1. Requires only 4 hrs.

Disadvantages:

1. Test is not simple

2. Equipment is complex

3. Cost is high

Dewar test

This test is similar to fosdick calcium dissolution test except that the final pH after 4 hrs is measured instead of the amount of calcium dissolved.

This test has not been adequately tested for clinical correlation.

Prediction of future caries activity based on previous caries experience

Action- as an alternative to chemical or bacteriological tests for determining caries activity, previous caries experience can be reasonable indication of future trends. It is better to omit occlusal surfaces in such estimates.

Equipment- dental light, mirror, explorer, radiographs.

Procedure:

Koch grouped 9-10yr old children into a high caries active group and a low caries active group on the basis of restored:

1. Proximal surfaces of incisors and first permanent molars.

2. Buccal surfaces of maxillary first permanent molars.

3. Lingual surfaces of mandibular first permanent molars.

Those who had a score of 4 or more were considered high caries active whereas those who had a 0 score were considered low caries active.

Drawbacks:

1. Caries will have already occurred in the population.

2. This method is not applicable to very young, when preventive intervention is desirable.

3. This method requires professional dental examination.

Yeast test

Salivary yeast are found to be related to the saliva flow rate and to the sex of the patient. Yeast are measured with a dip slide cultivation method known as ORICULT –N. The oral yeast concentration closely follow the DMF index values and that the presence of of

yeast in early childhood is a predictive of rampant caries in future. Infection with yeast can also be considered a sign of reduced salivary output which has

probably lasted for a lengthy period.

Some new caries activity tests

Cariostat

The Cariostat Saliva Buffer Test (CAT Buf Test), was developed to determine the buffering ability of saliva of an individual.

The person is asked to chew unflavored gum to stimulate flow of saliva, which is then collected by means of a small container.

An ample amount of saliva sample is poured into the Buf ampule containing  special indicators.

Color change is immediate and then compared to the standard reference color chart provided

The cariostat buffer saliva test

Caries risk test (CRT)

The buffering systems contained in saliva are capable of neutralizing acids that are harmful to teeth.

The extent to which the buffering systems can fulfill this important function in each case can be established by determining the buffering capacity of saliva.

The CRT buffer caries risk test allows the buffering capacity of saliva to be determined quickly and easily.

Advantages:

Quick and easy to conduct Results available after only five minutes Reliable results

Oratest

Oratest, a simple, economical, non-invasive and less time consuming test for estimating the oral microbial level.

Advantages:

1. It is an index for success of therapeutic measures and also helps to motivate and monitor effectiveness of educational programs relating to dietary and oral hygiene procedures.

2. No special instruments are required.

Limitations:

1. Lack of specificity as it does not identify the source of micro-organism.

Principle: is based on the rate of oxygen depletion by micro organisms.

Under aerobic conditions the bacterial enzyme, aerobic dehydrogenase transfers electrons or protons to oxygen.

Once oxygen gets utilized by the aerobic organisms and an anaerobic environment is attained, methylene blue [redox indicator] acts as an electron acceptor and gets reduced to leucomethylene blue.

The metabolic activity of the aerobic microorganism is reflected by the reduction of methylene blue to leucomethylene blue

The test is based on rinsing the mouth with sterile milk which dislodges the micro-organisms and also produces a substrate for their further metabolism. The formation of leucomethylene blue can be easily observed because of the white color of milk.

Cariogram

Cariogram was presented in 1996 by Bratthall to illustrate the interaction of caries related factors.

This model makes it possible to single out individual risk or resistance factors. The original cariogram was a circle divided into three sectors each representing factors

strongly influencing carious activity: diet, bacteria, susceptibility. Based on the concept of cariogram in 1997 bratthall et al developed an interactive caries risk

estimation model. The program operates in such a way that the individual data for a patient regarding bacteria,

diet, saliva and fluoride are entered into the program together with information regarding circumstances.

The values entered are based on the specific criteria. The score 0=most favorable and score 3=high unfavorable risk value

According to the built in formula the program presents a pie diagram where:

Bacteria: red sector-combination of amount of plaque and mutans streptococci. Diet: dark blue sector-combination of diet contents and diet frequency. Susceptibility: light blue sector-combination of fluoride program, saliva secretion and saliva

buffer capacity. Circumstances: yellow sector-combination of caries experience and related diseases. Chance of avoiding caries: green sector. Thus cariogram model is a simple way to illustrate how caries related factors can interact. It is useful in situations when there is a need to discuss the importance of etiologic factors.

Limitations of caries activity tests

None of the caries activity tests are highly reliable as indicators of expected caries increments. This is because these tests measure a single parameter such as acid produced or colony counts

of a bacterial species. Dental caries is a multifactorial disease and caries predictive tests do not encompass those

factors involved in determining caries resistance such as fluoride exposure, maturation of enamel, or immune protection.

The limitations inherent in a single caries activity test are clear. And this is the reason why the best predictor of expected caries activity can be achieved from

the combined use of several selected tests.

Caries activity tests:principle underlying test method of quantitation and clinical significance

REFERENCES

1. Newbrun E, Cariology; 3rd edition: pg 273-291.

2. Harris N.O, Primary Preventive Dentistry; 6th edition: pg 337-362

3. Axelsson P, diagnosis and risk prediction of dental caries; vol 2: pg 151-178

4. SS Hiremath, Textbook of preventive and community dentistry; pg 329-334

5. Peter S, Essentials of Preventive and Community dentistry; 3rd edition. Pg 359-367

6. Bratthall D, Hansel Petersson G. Cariogram- a multifactorial risk assessment model for a

multifactorial disease. CDOE 2005;33:256-64

7. S. Bhasin, P. Sudha, T.Anegundi R. Chair side simple caries activity test: oratest. JISPPD

2006:24:76-79