PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
NAME OF THE CANDIDATE AND ADDRESS
Dr. RESHMA NAIK
MARATHA MANDAL’S NATHAJIRAO G HALGEKAR INSTITUTE OF DENTAL SCIENCES AND RESEARCH CENTRE, R.S.NO.47A/2, NEAR K.S.R.P GROUND, BAUXITE ROAD, BELGAUM-590 010.
NAME OF THE INSTITUTION
MARATHA MANDAL’S NATHAJIRAO G HALGEKAR INSTITUTE OF DENTAL SCIENCES AND RESEARCH CENTRE, BELGAUM
COURSE OF STUDY AND SUBJECT
MASTER OF DENTAL SURGERY IN PERIODONTOLOGY
DATE OF ADMISSION TO COURSE
TITLE OF TOPIC
DETECTION OF SPIROCHETES AND TREPONEMA DENTICOLA IN PERIODONTAL HEALTH AND DISEASE BY COMPARING MICROSCOPY AND POLYMERASE-CHAIN REACTION TECHNIQUES – AN INVITRO STUDY
Brief resume of intended work :
6.1 Need for study :
Periodontal disease is an ecogenetic multifactorial disease, characterized by interaction between pathologic bacteria and the host defense mechanism which eventually leads to periodontal destruction and tooth loss.
The Oral Spirochetes are often the dominant bacterial types observed in diseased periodontal sites and yet they are less studied and understood members of plaque flora.1
This dearth of knowledge relative to oral spirochetes reflects the difficulty in isolating and cultivating;1
- In cultivation methods Spirochetes being relatively fastidious
barely showed up in count data.2
- As spirochetes are delicate organisms relative to other bacterial
types found in dental plaque, this affects their isolation as they are
Also no systematic studies have been conducted on association of spirochetes in periodontal diseases in India. And as the prevalence of microorganisms vary depending on the geographical distribution, food habits and immune response of an individual/population, hence the need of the study.
6.2 Review of Literature:
G C Armitage, W R Dickinson, R S Jenderseck, S M Levine, & D W Chambers (1982):- studied the relationship between the percentage of subgingival spirochetes and severity of periodontal disease. The correlations between various clinical assessments of inflammatory periodontal disease and percentage of motile bacteria in subgingival flora of sites representing widely varying states of periodontal disease was studied using Dark field microscopy and they found, Clinically healthy sites harboured much lower percentage of motiles than did clinically diseased sites, also percentage of spirochetes increased with greater severity of periodontal disease. And most of the observed variation in the percentage of motile bacteria could be accounted for by variations in the percentage of spirochete.3
Lloyd G Simonson, Charles Goodman, John J Bial and Harold E Morton (1988):- Estimated quantitatively, relationship of Treponema denticola to severity of periodontal disease. They presented the first quantitative evidence of a positive relationship between a specific spirochete and the morbidity of periodontitis. The specific spirochete found was serotype of Treponema denticola.
A biotin-avidin ELISA procedure was developed with a monoclonal antibody specific for a serovariety of Treponema denticola. They concluded that Treponema denticola was present at significantly elevated levels in plaque samples collected from deep-pocket sites of patients with severe periodontitis relative to healthy control groups with moderate disease.4
A Coffey, W A Coulter, G J Linden(1995):- conducted a study to compare the feasibility of direct microscopy using light silver stain with dark field microscopy for differential counting of subgingival plaque from patients with untreated adult periodontitis. They found differential counts of plaque morphotypes assessed by both methods showed close agreement and the proportions of spirochetes assessed by both methods were significantly associated with probing depth. And they found silver stain proved to be a simple, rapid and inexpensive method for differential counting of plaque composition.5
Annette Moter, Carina Hoenig, Bong-Kyu Choi, Birgit Riep, Ulf B Gobel (1998):- Conducted a study on molecular epidemiology of oral treponemes associated with periodontal disease. To assess the role of spirochetes, oligonucleotide probes were designed for the identification of both cultivable and so far uncultivable spirochetes in periodontitis patients. The plaque samples were submitted to direct in situ hybridization or dot blot hybridization after prior amplification with eubacterial primers. They found all treponemes described here predominated at diseased sites but were detected only infrequently at periodontally healthy sites, indicating that they indeed may be of etiologic relevance.6
Dewhirst F E et al. (2000):- studied the Diversity of periodontal spirochetes by 16s rRNA analysis. The purpose of this study was to examine the diversity of spirochetes in the subgingival pocket of multiple subjects with a range of periodontal conditions, including 2 healthy, 1 adult periodontitis, 3 acute necrotizing ulcerative gingivitis, 8 refractory periodontitis and 1 HIV periodontitis.
The 16s rRNA genes of spirochetes in plaque were amplified by PCR using spirochete selective primers. A total of 542 clones were sequenced and subjected to phylogenetic analysis to establish the diversity of human oral spirochetes. The sequences were clustered into 10 known cultivated Treponema species and into 47 as yet uncultivated Treponema species. Of the cultivable species, Treponema Denticola, Treponema. Maltophilum and Treponema species Smibert-3 were most commonly encountered in diseased subjects but rarely in healthy subjects.7
Claude Bayingana, Ashley Pretorius and Charlene W J Africa (2010):- conducted a study on Comparison of PCR and BANA hydrolysis in detecting Oral Anaerobes in subgingival plaque. The objective of this study was to evaluate the sensitivity and specificity of BANA and PCR for detecting oral anaerobes such as P.gingivalis, Tannerella Forsythia and Treponema Denticola ( red complex)
Of 372 samples analysed 7.25% tested positive for BANA test and 36.29% yielded a positive PCR test. Thus they concluded that PCR is more sensitive than BANA for detecting small numbers of members of red complex( P.gingivalis, T.forsythia and Treponema denticola) in plaque samples and it has an advantage over BANA in that it has the ability to accurately detect and identify specific species even when they are present in low numbers.8
6.3 Objectives of the study:
1. To detect the Prevalence of Total Spirochetes in patients with healthy periodontium, gingivitis and periodontitis using Microscopy and PCR techniques.
2. To detect the Prevalence of Treponema Denticola in patients with healthy periodontium, gingivitis and periodontitis using Microscopy and PCR techniques.
3. Comparison of Microscopy (Silver nitrate staining) technique and PCR technique in detection of Spirochetes and Treponema Denticola
Methods and Materials
7.1 Source of data:
The subjects for sample collection consists of patients visiting Department of Periodontology and samples will be analysed at Department of Molecular Biology and Immunology at Maratha Mandal’s NGH Institute of Dental Sciences and Research Center, Belgaum.
7.2 Methods of collection of data:
150 subjects will be selected and divided into 3 groups of 50 subjects each:-
1) Healthy group 2) Gingivitis group 3) Periodontitis group
1)Healthy group:- Probing depth ≤ 3mm, no bleeding on probing, and
No clinical signs of inflammation
2)Gingivitis group:- Probing depth ≤ 3mm, Bleeding on probing and
Gingival index (Loe H & Sillness J 1963) and Plaque index (Sillness & Loe 1964) will be recorded in all subjects. Written informed consent will be taken from all subjects.
i) Patients with any systemic diseases/ medically compromised patients
ii)Patients who have received - periodontal therapy,
antibiotics/antimicrobials within 3 months prior to sampling
iv) Pregnant women and Lactating mothers
v) Tooth with Cervical/Proximal/Subgingival Caries or Restorations
Plaque samples shall be collected from subgingival sites in healthy, gingivitis & periodontitis groups using sterile curettes and transferred to lab in an appropriate transport media. The Prevalence of Spirochetes and Treponema Denticola is first detected by Microscopy (Silver nitrate staining) technique, and then followed by Pan spirochetes specific PCR and Treponema Denticola specific PCR techniques.
Sensitivity and Specificity of Microscopy and PCR techniques shall be compared.
7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals?
YES. This study will require the collection of sub gingival plaque
from the patients
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
List of References: 1) W J Loesche. The Role of Spirochetes in Periodontal disease. Adv Dent Res 1988;2:275-83.
2) Richard P, Ellen & Vaia B, Galimanas. Spirochetes at the forefront of periodontal infections. Periodontology 2000;38:13-32.
3) G C Armitage, W R Dickinson, R S Jenderseck, S M Levine, D W Chambers. Relationship between the percentage of subgingival spirochetes and the severity of periodontal disease. J Periodontal 1982;53:550-56.
4) Lloyd G Simonson, Charles H Goodman, John J Bial, Harold E Morton. Quantitative relationship of Treponema denticola to severity of periodontal disease. Infect Immun 1988;56:726-28.
5) Coffey A, Coulter W A, Linden G J. A feasibility study on the use of direct light silver stain compared with dark field microscopy for differential counting of subgingival plaque. J Periodont Res 1995;30:342-48.
6) Annette Moter, Carina Hoenig, Bong-Kyu Choi, Birgit Riep, Ulf B Gobel. Molecular epidemiology of oral treponemes associated with periodontal disease. J Clin Microbiol 1998;36:1399-403.
7) Dewhirst F E, Tamer M A, Ericson R E, Lau C N, Levanos V A, Boches S K et al. The diversity of periodontal spirochetes by 16S rRNA analysis. Oral Microbiol Immunol 2000;15:196-202.
8) Claude Bayingana, Ashley Pretorius, Charlene W J Africa. Comparison of PCR and BANA hydrolysis in detecting oral anaerobes in subgingival plaque. Afr J Microbiol Res 2010;4:771-74.