EVALUATION OF RED COMPLEX ORGANISMS AND

SALIVARY pH IN HEALTH, GINGIVITIS AND CHRONIC PERIODONTITIS –

A CLINICO-MICROBIOLOGICAL STUDY

Presented by: Dr Harshavardhan PatwalGuided by: Dr. Nandini Manjunath

Prof & H.O.D Dept of Periodontics

A. J. Institute of Dental Sciences, Mangalore

INTRODUCTION

• Periodontal diseases are multifactorial infections elicited by a complex of bacterial species that interact with host tissues and cells causing the release of broad array of inflammatory mediators which lead to distruction of periodontal structures.

• The ecological factors provided by the environment of the oral cavity are directly proportional to the species richness and species biodiversity of the microorganisms that reside on the teeth.

INTRODUCTION

• The main ecological factors are pH, saliva, temperature and redox reactions .

• The majority of the organisms prefer neutral pH levels (pH7). Saliva acts as a buffer maintaining the pH in the oral cavity between 6.75-7.25.

OBJECTIVES OF THE STUDY:

1.To estimate the level of salivary ph in healthy, gingivitis, chronic periodontitis patients.

2. To estimate the “Red Complex” organisms in healthy, gingivitis, chronic periodontitis .

MATERIALS AND METHODS

• Study sample consist of 60 adult subjects (both male and female) of 35-60 years of age.

• A brief case history was recorded and an informed consent was taken for all the patients.

• Clinical examination was recorded and study subjects were divided into 3 groups ( healthy, gingivitis, chronic periodontitis) based on the objectives.

INCLUSION CRITERIA

• Patients with healthy periodontium was considered as a control group.

• Subjects with 30% periodontal pockets with probing depth of equal to or more than 5mm in each quadrant .

• Patients who have not received any antimicrobial therapy for the last 6 months.

EXCLUSION CRITERIA:

• Inability to provide information or cooperate to dental examination

• Inability to accept periodontal treatment

• Patients diagnosed with diabetes mellitis, cardio-vascular or kidney diseases or any nerve condition for which prophylactic antibiotic treatment before the dental examination is necessary.

• Smokers and individuals who consume alcohol.

• Pregnant and lactating women , women taking oral contraceptives .

• Malignancy.

CLINICAL PARAMETERS:

• Complete periodontal examination was done using the parameters such as plaque index (sillness and loe) and gingival index (loe and sillness) probing pocket depth and clinical attachment level.

• Red complex organisms are assessed using BANA test.

• Salivary ph was measured using a Digital pH meter (systronics MK-6).

COLLECTION OF SALIVA :

• Subjects were instructed not to eat or rinse within 60 minutes prior to sample collection. Whole saliva was collected simply by drooling into a sterilized vial with the forward tilted head or by allowing the saliva to accumulate in the mouth and then expectorate into a vial.

• The resulting saliva was stored in at -20°C until the determinations are performed.

Unstimulated saliva is collected by tilting the head of the patient downward and collecting it in the cup .

WORKING OF pH METER PRINCIPLE • Measurement of voltage difference between 2

electrodes placed in a solution .

PARTS OF pH METER

1. Calomal electrode :external reference electrode whose electrical stimulation depends on test solution .

2. Glass electrode

3. Electronic meter

PARTS OF ELECTRONIC METER

1.Calibration key : to enter the calibration of known solution.

2.Confirmation key To confirm the calibraton value

3.Arrow keys : manually select pH of buffer .

4.MR – recall stored value

5.Memory key : to store in memory .

REFERENCE ELECTRODE WITH GLASS ELECTRODE IS CALLED

COMBINATION ELECTRODE

CALIBRATION OF PH METER :

• Step 1 – wash electrode with distilled water .• Step 2- calibrate pH meter with pH 7.0 buffer • Step 3 – when ready blinks on the screen press

confirmation key • Step 4 – again rinse the electrode with distilled water • Step 5 – place the electrode in pH of 4.0 • Step 6 – press calibration key • Step 7 – check the pH of test solution .

CALIBRATING SOLUTIONS

DETERMINATION OF “RED COMPLEX” ORGANISMS :

• BANA test is a enzymatic chair side test. It is a modern chair side para-clinical method designed to detect the presence of one or more anaerobic bacteria ,commonly associated with periodontal diseases.

• This test is very sensitive detecting small quantities of pathogens, no meaningful diffrences could be found between DNA probes. Immunological reagents and BANA test.

PRINCIPLE OF BANA TEST :• Peptides of the 3 bacterial “Red Complex” species

(T.denticola,P.gingivalis,B.forsythus) can hydrolyse the peptide analog N-benzoyl DL-Arginine- napthalamide. One of the hydrolytic products of this reaction is B-naphthylamide, which reacts with a reagent, which is imbedded in the upper strip of the test, producing a permanent blue color .

• Blood and saliva do not interfere with the test .

DIRECTIONS OF USE :• Anaerobic microorganisms associated with periodontal disease

are found in the subgingival plaque. To obtain specimens for testing, sites should be cleared of supragingival plaque.

• A Gracey curette is used to obtain subgingival plaque specimens , which are placed on the lower matrix. Before taking another specimen, wipe the curette on a clean piece of cotton or other suitable wipe to prevent carry-over of plaque.

• The upper matrix is moistened with saline solution and the test is folded so as the two matrices are coming in contact. It is incubated for 5 minutes at 55 Celsius degrees temperature. If BANA positive species are present when the test is opened, a permanent blue coloration on the upper matrix is found . The higher the concentration of bacterial species, the darker blue coloration is present on the test. According to the result, the test can be positive, weak positive, or negative .

BANA STRIP:

COLLECTION OF SUBGINGIVAL PLAQUE :

BANA KIT

INCUBATING STRIP FOR 5 MINUTES UNTIL THE BEEP SOUND .

COMPARASION OF TEST RESULTS

STATISTICAL ANALYSIS

• Statistical analysis was carried out using chi square tests and fishers exact test .

COMPARISON WITH pH AND BANA

CrosstabBANA Total

NEGATIVE WEAKLY POSITIVE

POSITIVE

pH 6.26-7.25 Count 2 3 0 5% within BANA

66.7% 42.9% 0.0% 33.3%

6-6.25 Count 1 4 0 5% within BANA

33.3% 57.1% 0.0% 33.3%

5.5-5.9 Count 0 0 5 5% within BANA

0.0% 0.0% 100.0% 33.3%

Total Count 3 7 5 15% within BANA

100.0% 100.0% 100.0% 100.0%

HERE IT IS NOTED THAT SIGNIFICANTLY LOWER PH WHEN BANA IS POSITIVE. ALL 5 CASES WHERE BANA WAS POSITIVE THE PH WAS <5.9 P VALUE 0.001. THIS IS

SIGNIFICANT AT THE 5 % ERROR MARGIN.

Chi-Square Tests

Value P VALUE

Fisher's Exact Test 13.114 .001

N of Valid Cases 15

CONCLUSION :• There is a positive correlation, both clinically and

statistically, between the BANA test results and the pH seeing the current stage of periodontal destruction. The BANA test results are not correlated with the degree of oral hygiene evaluated against the plaque index, so the quality and not quantity of bacterial plaque influence the test results.

• Among these possibilities, the microbial-enzymatic BANA test is a quick, chair-side test with a very good sensibility, giving the clinician details about the microbial composition of the subgingival plaque and consequently about the clinical evolution of the periodontal disease. BANA test is also offering therapeutic orientation regarding the need for antimicrobial therapy.

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