Brief Resume of the Intended Work Need for the Study

Brief Resume of the Intended Work
Need for the Study:

Microbial & clinical studies have revealed that, periodontitis is caused by bacteria, which inhabitat periodontal pocket & bacterial colonization may play an important role in pathogenesis of the disease.^1,2

A high proportion of progressing periodontal infection in adults have been associated with presence or elevated levels of Porphyromonas gingivalis & that it might not be a normal inhabitant of periodontally healthy subjects^1,2,3. Facilitating detection of this pathogen would be a major benefit in periodontal risk assessment⁴.

The Culture & DNA hybridization is currently available for testing the presence of periodontal pathogens , the culture technique is advantageous in detecting the antibiotic sensitivity of different pathogens in order to determine the appropriate therapy & disadvantage being, time consuming for growth & isolation of specific organisms, survival of bacteria during transportation to laboratory is questionable. DNA probes is used for identification of specific pathogens regardless of their viability, disadvantage being lesser limit of sensitivity.^2,5

In recent years, based on molecular biology techniques ,Polymerase chain reaction(PCR) has been developed which is simple, sensitive, less time consuming technique. PCR replicates a fragment of RNA or DNA for the detection of specific periodontopathic bacteria. It utilizes a pair of primer, that are selected from opposing strands of a gene which is specific to a particular organism.^2,4,5

The purpose of the present study is to detect Porphyromonas gingivalis using Polymerase chain reaction(PCR) in periodontally healthy subjects & chronic periodontitis patients.

Objectives of the study:

1. 
   The objective of this study is to detect Porphyromonas gingivalis in subgingival plaque samples in healthy subjects & chronic periodontitis patients using polymerase chain reaction technique .
   

2. 
   To assess the relationship of Porphyromonas gingivalis in health & chronic periodontitis .
   

The following observations will be made

a).Presence of Porphyromonas gingivalis in 10 clinically healthy periodontium.

b) Presence of Porphyromonas gingivalis in 10 chronic generalized periodontitis patients from subgingival plaque samples obtained from a single site of pocket depth measuring ≥5mm per patient.

Materials and Methods
1. Source of data: Systemically healthy patients visiting the department of Periodontics, Krishnadevaraya College Of Dental Sciences & Hospital, Bangalore, will be selected.

-Clinically healthy periodontal subjects(control group)

-Chronic generalized periodontitis patients (test group)
2. Method of collection of data:
Sample size: 20 patients attending the out patient department of periodontics, KCDS, Bangalore and satisfying inclusion and exclusion criteria will be taken for the study
Control group – 10 systemically healthy patients with clinically healthy periodontium and

Test group -10 systemically healthy patients with history of chronic generalized periodontitis , satisfying the inclusion criteria .

1 subgingival plaque sample from one site of pocket depth measuring ≥5mm from each chronic generalized periodontits patients.

Inclusion criteria
Control Group

• 
  Healthy subjects in the age group of 25-50yrs
  
• 
  Systemically healthy subjects with clinically healthy periodontium
  

Test Group

• 
  Patients with history of chronic generalized periodontitis who are systemically healthy with age group 25-50yrs
  
• 
  Patients with a minimum of 4 periodontal pockets with ≥5mm
  
• 
  Patients who are willing to co-operate with the study protocol
  

Exclusion criteria:
- Medically compromised patients

- Patients who have received any periodontal therapy surgical or non surgical , during

past 6 months of baseline examination

- Use of antibiotics in the past 6 months

- Patients with history of any habits like smoking, alcohol, pan chewing
3. Sample Collection

A randomized sampling method is used for selecting 20 patients, out of which 10

patients with the history of chronic generalized periodontitis(test group)& 10 subjects

with a clinical healthy periodontium (control group) will be be selected. After obtaining

an informed consent from the patients,

Data will be recorded for

1. 
   Plaque index – using Silness & Loe 1964(Annexure 1)
   
2. 
   Gingival index – Loe & Silness 1963(Annexure 2)
   
3. 
   Periodontal pocket depth – measured using UNC –15 probe
   

Following clinical examination, one site with pocket depth measuring ≥5mm will be

selected in chronic generalized periodontitis patients & one site with clinically healthy

periodontium will be selected in healthy subjects. The sampling site are isolated using cotton rolls.

Supragingival plaque is removed using cotton roll & then air dried. Subgingival

plaque samples are collected using a sterile endodontic paper point no 25 inserted into

the pocket until resistance is felt . After 30 seconds, paper point is removed.
The collected sample is immediately suspended in phosphate buffer saline. The samples

are stored at 0 degree till taken for PCR analysis.

4. Statistical analysis
The statistical difference between the presence of Porphyromonas gingivalis in health

subjects & chronic periodontitis patients is detected by ANOVA ( Analysis of variance ) test

List of References
1) Hui Wen Yang, Yu-Feng Huang & Ming-Yung Chou – Occurrence of Porphyromonas gingivalis & Tannerella forsythensis in periodontally diseased & healthy subjects J Periodontol 2004;75(8);1077-1083
2) K. Watanabe & T.O. Frommel - Detection of Porphyromonas gingivalis in the oral plaque samples by use of the polymerase chain reaction J.Dent.Res1993;72(6); 1040-1044
3) Marlise.I.Klein & Reginaldo.B.Goncalves -Detection of Tannerella forsythensis & porphyromonas gingivalis by Polymerase chain reaction in subjects with different periodontal status. J Periodontol 2003; 74(6);799-802
4) Yasuo Takeuchi , Makoto Umeda, Mitsuo Sakamoto, Yoshimi Benno, Yi Huang, Isao Ishikawa - Treponema sockranskii, Treponema denticola & Porphyromonas gingivalis are associated with periodontal tissue destruction.

J Periodontol 2001;72(6) 1354-1363

5) Keiko Watanabe & Thomas O Frommel – Porphyromonas gingivalis,

Actinobacillus actinomycetemcomitans & Treponema denticola in oral plaque

samples using PCR. J Clin Periodontol 1996; 23; 212-219

6) Riggio MP, Macfarlane TW, Mackenzie D, Lennon A, Smith AJ, Kinane D –

Comparison of polymerase chain reaction & culture methods for detection of actinobacillus actinomycetemcomitans & porphyromonas gingivalis in subgingival plaque samples

. J. Periodont res 1996;317;496-501
7) Yoshihiko Kasuga, Kazuyuki Ishihara & Katsuji Okuda - Significance of detection of Porphyromonas gingivalis, Bacteriodes forsythus, & Treponema denticola in periodontal pockets. Bull Tokyo dental coll 2000; 41(3);109-117
8) Basak Dogan, Johanna Antinheimo, Deniz Cetiner, Aysen Bodur, Gulnur Emingil, Eralp Buduneli et al-Subgingival microbiata in Turkish patient with periodontitis . J Periodontol 2003 ;74(6) 803-814