CARIES ACTIVITY TESTS

  CARIES ACTIVITY :-Refers to the increment of active lesion over a stated period of time i.e. it’s a  
measure of speed of progression of caries lesion

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   CARIES SUSCEPTIBILITY:-Refers to the inherent tendency of host and target tissue, the tooth , to be afflicted by the caries process i.e. Susceptibility of tooth to a caries producing environment

   CARIES ACTIVITY TEST :- Measures the degree to

  which the local environment challenge favours the probability of carious lesion

USES:
1.  To determine the need and extent of           personalized preventive measures

2.  To serve as an index of success of           therapeutic measures

3.  To motivate and monitor the effectiveness of   education progress

4.  To manage progress of restorative               procedures

5.  To identify high risk individuals

1. LACTOBACILLUS COLONY COUNT TEST

  Introduced by HADLEY in 1933
  PRINCIPLE:-
  Estimates the number of cariogenic and aciduric   bacteria in patients saliva on peptone agar plate.(pH. 5.0)  PROCEDURE
  Obtain early morning saliva (after paraffin) 
  Dilute 1:10 and  1:100 concentration
  0.4 ml of each dilution is spread on surface of Rogasa’s   SL agar plate
  Plates incubated for 3-4 days at 37⁰ c
  Count the number of LBS colony using colony counter.

RESULT

  ADVANTAGES
  1.Monitors the effectiveness of restorative dentistry
  2.Simple to carry out
  3.Useful as screening test

    DISADVANTAGES
  1. Inaccurate to predict the caries on set
  2. Does not exclude the growth of other aciduric
        organism
  3. Counts not reliable
  4. Results not available for several days
  5. Counting is a tedious process

       
  2. CALORIMETRIC SNYDER TEST

PRINCIPLE:- (By Snyder in 1951)
It measures the ability of salivary micro- organisms to form organic acids from carbohydrate medium
Indicator used –  Bromocresol green. It changes colour from  green to yellow in pH range of 5.4 to 3.8.
 
PROCEDURE: –
0.2 ML of stimulated saliva collected mixed with 10 ml melted agar, solidify and incubate at 37⁰c,  amount of acid produced is detected by change in the pH indicator and compared to control tube after 24,48,72 hrs

Colour Observations in Snyder test

24 Hoursà              48 Hoursà             72 hours
_____________________________________________________If yellow                   If yellow                   If yellow_____________
marked caries         definite caries          limited caries
susceptible              susceptibility           susceptibility
___________________________________________________
If green                   If green                    If green_____________
continue to   continue to   caries inactive
incubate &   incubate &
observe at 48   observe at 72 hrs.
hrs .

  ADVANTAGES
  1.  Simple
  2.   Valuable in assessing the oral cariogenic   challenge
  3.  Only one tube, no serial dilutions required
 
  DISADVANTAGES
  1.  Time consuming    
  2.  Sometimes colour changes not so clear

  3. SWAB TEST

  PRINCIPLE:- (Grainger et al 1965)
  Same as Snyder test.
  Oral flora sampled by swabbing buccal surfaces of teeth   which is incubated.
  The change in pH following 48 hrs.

  RESULT
  pH 4.1 and < 4.1       = marked caries activity
  pH 4.2 to 4.4             = active
  ph 4.5 to 4.6              = slightly active
  pH 4.6 and over        = caries active

  ADVANTAGES
  1. Used in children with low or no caries   experience
  2. No collection of saliva is required

4. STREPTOCOCCUS MUTANS LEVEL IN SALIVA
 
PRINCIPLE :-
Measures the number of S. mutans colony forming units, per unit volume of saliva and culturing of plaque samples from discrete sites. Medium for incubation of Mitis salivarius agar with high concentration sucrose and 0.2 micron bacterium per ml (MSB) to suppress non S.mutans colonies.
PROCEDURE: –
 Sample obtained by use of tongue blades which are then pressed against Streptococcus mutans selective MSB (Mitis Salivarius Bacitracin ) Agar in Petridish incubate at 37⁰c for 48 hrs in 95 % at 5% co₂ gas mixture

INTERPRETATION

* Level of S mutans more than 10⁵ /ml of saliva = unacceptable
* Level of S mutans 4.5 x 10⁴ per ml for smooth surface and 10³ per ml for occlusal fissure

ADVANTAGES
 Can be used as an adjunct in caries management

DISADVANTAGES
1.Difficult to distinguish between carrier   state   and cariogenic infection
2.Constitutes less than 1% of total oral flora
3.Mutans tend to be located at specific sites

5. DIP SLIDE METHOD FOR S. MUTAN’S COUNT

 PRINCIPLE:-
Estimates level of S mutans in saliva
 PROCEDURE: –
Stimulated saliva is poured on a slide coated with MSA containing 20% sucrose
Moisten the agar and drain excess saliva
2 disc’s containing 5 mg of bacitracin are placed on agar 20 mm apart
Slide is closed and incubated for 48 hrs. at 37⁰c in a sealed candle jar

INTERPRETATION: –

SCORE 1=LOW
Colonies are discrete and counted at 15 x magnification. Total count of CFU inside inhibition zone <200

SCORE 2= MEDIUM
Colonies are discreet and number in zone of inhibition is >200 and 32 x magnification

SCORE 3= HIGH
Colonies are tiny and cover the inhibition zone .
The number of colonies uncontrollable with 32 x magnification

6. SALIVARY BUFFER CAPACITY TEST

PRINCIPLE:-
It measures the ml of acid required to lower the pH of saliva through an arbitary pH interval.

PROCEDURE: –
10 ml of stimulated saliva (after food) is collected under oil 5 ml is added to beaker.
Correct the pH meter to room temperature .
Adjust pH of saliva to 7 by adding lactic acid or base.
Lactic acid is added to sample until pH of 6 is reached.
Number of ml needed to reduced pH from 7 to 6 is measure of buffer capacity.

EVALUATION

There is an inverse relation between buffering capacity and caries activity.

ADVANTAGES
Simple to carry out

DISADVANTAGES
Does not correlate with caries activity

7. ALBANS TEST
  ADVANTAGES
  1. Simplicity 
  2. Low cost
  3. Diagnostic
    
   PROCEDURE
 1.  60 gms of Snyder test agar is placed in 1 ltr. of   water and   boiled over low flame
 2.  agar is distributed 5ml per test tube
 3.   allowed to cool
 4.  patient asked to expectorate small amount of saliva in test   tube
 5.  test tube is incubated at 98.6 F up to 4 days
 6.   observe tube for color change daily and   depth in medium   to which change was seen

SCALE FOR SCORING
1. No colour change = ¾
2. beginning colour change = +
3. ½ colour change = + +
            (from top down)
4. ¾ colour change = +++
          (from top down)
5. total yellow =++++

  8. FOSDICK TEST

PRINCIPLE:-
Measures the mg of enamel dissolved in 4 hrs by acid formed when saliva is mixed with glucose.
 PROCEDURE: – 
25ml of stimulated saliva is collected the remaining is placed in 8 inch sterile tube with 0.1 gram of   powdered enamel.A tube is sealed and shaken for 4 hours at body temperature. Analyze calcium content. Amount of dissolution increases as caries activity increases.

  ADVANTAGES:-
  1.Correlation good


  DISADVANTAGES:-
  Test complicated,
  Complicated equipment
  Expensive
  Trained person required.

9. STREPTOCOCCUS MUTANS SCREENING TEST

  A. Plaque/tooth pick method (MSA agar)
  B. Saliva/Tongue Blade method (MSB agar)

DENTAL CARIES VACCINE

History of vaccines:

– Edward Jenner was the pioneer in the field of immunization. He developed a vaccine against small pox.



– Louis Pasteur succeeded in developing vaccine against anthrax and rabies.

The advent caries vaccines

Target organisms •Lactobacillus •Streptococcus mutans

  Lactobacillus was used as an antigen. Immunization against lactobacillus was only partially successful and could not provide adequate protection against caries, this was because it was more of a consequence than cause of caries initiation and was present only in deep carious.

  Hence, streptococcus mutans became the target in virtually all immunization experiments after their redetection in 1960.

Prevention of Dental Caries by Immunization:

  A vaccine could be administered before the deciduous dentition has erupted (6 mths of age) it would prevent the disease in the children, who show the greatest incidence of caries.

 The vaccine could be given at the same time as that against diphtheria and tetanus.

   Individual parents would make the decision whether to have their child vaccinated the principle of individual freedom would not be violated.

Mechanism of action of caries vaccine:

  1. Active immunization / vaccination:

–      Vaccination involves administration of antigen into the host, stimulating host immune response to produce antibodies against the antigen.

        Either whole antigen or subunit vaccines can be administered.      

        The disadvantage of this is that the antigen may contain the fimbrial M protein, which cross reacts with heart tissue.

  Active immunization can be administered either to stimulate systemic antibody production or mucosal antibody production.

 Advantage of active immunization is that it results in enhanced immune responses with possibilities of longer periods of protection.

Disadvantage is that it cannot be used in immuno compromised individuals.

  2.Passive immunization:

   In this route, ready-made antibodies against streptococcus mutans antigens are isolated and administered to provide immediate protection against caries.

It can be achieved in the following ways.

A.  Local Route

B.  Systemic Route

Routes of administration:

In general, routes of immunization with streptococcus mutans are:

i)     Oral

ii)    Systemic (subcutaneous)

iii)   Active gingivo-salivary

iv)   Intranasal

v)    Tonsillar

vi)    Minor salivary gland

vii)   Rectal

Oral Route of Administration:

Here the antigen is applied by oral feeding, gastric intubation or in form of vaccine containing capsules of liposomes.

Disadvantages: •Not ideal for reasons including detrimental effects of stomach acidity on antigen. •Inductive sites are relatively distant. •The rise in secretory antibodies produced was of short duration. •The immunological response initiated is predominantly by IgA antibodies.

Intranasal Route of Administration:

More recently attempts have been made to induce protective immunity in mucosal inductive sites that are in closer anatomical relationship to the oral cavity.

Intranasal installation of antigen targets the nasal-associated lymphoid tissue (NALT).

Tonsillar Route of Administration:

Tonsillar tissue contains elements of immune induction of both secretory IgA and IgG responses.

Palatine tonsils especially the naso-pharyngeal tonsils have been known to contribute precursor cells to mucosal effector sites such as salivary glands.

Minor salivary gland route of administration:

– Minor salivary glands that populate the lips, cheeks, and soft palate can be potential routes for mucosal induction of salivary immune responses.

Advantages: –Lower doses of antigen are required as degradation of the antigen by proteolytic enzymes and de-naturated by acid is lower than the GIT. –Induces both systemic and mucosal immunity –

– Administration is relatively easy.

Rectal route of administration: – More remote mucosal sites have also been investigated for inductive potential. – Colorectal region as an inductive site has been suggested because of the fact that this site has the highest concentration of lymphoid follicles in the lower intestinal tract.

– Thus use of vaccine suppositories as one alternative for children in whom respiratory ailments preclude use of intranasal application of vaccine.

Subcutaneous route of administration: • It was used successfully in animal trials and elicited predominantly serum IgG, IgM, and IgA antibodies. • Transport of antibodies, complement, sensitized lymphocytes access via the blood supply to the gingiva and then through the junctional epithelium into crevicular fluid. §

Disadvantages:

May induce antibodies to heart muscle

 Topical gingivo salivary immunization:  

– This route has been exposed in order to exclude any potential systemic side effects and to localize the immune response to the oral cavity.

– A prerequisite for successful gingival immunization was preparation of a smaller molecular weight streptococcal antigen, as high molecular weight antigen can’t penetrate the crevicular epithelial barrier.    

Barriers to use of vaccine:

–  Challenge presented by complex diet.

–  Shift to other cariogenic serotypes or organisms

–  Cross-reaction with heart tissue.

–  Dental caries is not considered a life threatening disease to warrant the use of a vaccine for its prevention.

– Dental caries is considered a multi-factorial disease of which microorganisms are only one of the various factors.

Recent advances in Caries Vaccine production:

1. Sub-unit vaccines

2. DNA vaccines

3. Adjuvant – Cholera toxin, Salmonella toxin

4. Liposomes

5. Biodegradable micro spheres

6. Bio-adhesives

7. Plantigens and Plantibodies

8. Advances in route and time of vaccine administration.

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