COMPARISON OF CULTURE & POLYMERASE CHAIN REACTION IN THE IDENTIFICATION OF TANNERELLA FORSYTHIA IN PERIODONTAL HEALTH & DISEASE AN IN-VITRO STUDY
Brief resume of intended work : 6.1 Need for study :
The human oral cavity is a highly complex ecosystem inhabited by an estimated 500 bacterial species, majority of which are commensals.1 A few like Porphyromonas gingivalis and Tannerella forsythia have been implicated as important pathogens in adult periodontitis.
Tannerella forsythia is a non-motile, spindle shaped, highly pleomorphic rod and a gram negative obligate anaerobe. A high proportion of periodontal infections in adults have been associated with the presence, or elevated levels of Tannerella forsythensis. The presence of this specific putative pathogen combined with other clinical characteristics, can indicate future risk of disease and the level of aggression. Facilitating the detection of these pathogens would hence, be a major benefit in periodontal assessment.
Inspite of the overwhelming evidence implicating Tannerella forsythia in the pathogenesis of periodontitis, this bacterium remains an understudied organism. No systematic studies have been conducted on association of Tannerella forsythia in periodontal diseases on Indian population, hence the need of the study.
6.2 Review of Literature : Tannerellaforsythia, first isolated at The Forsyth Institute from subjects with progressing advanced periodontitis in the mid-1970s was described as ‘fusiform Bacteroides’ by Tanneret al.2Around the same time, Tannerella forsythia was isolated as one of the Bacteroides group from the extensive cultural studies of periodontal infections by Moore and Holdeman-Moore at the Anaerobe laboratory of the Virginia Polytechnic Institute.3 Eugene J.Leys, Sharon R Lyons, Melvin L Moeschberger et al (2002) conducted a study to examine the relationship of the presence of Bacteroides forsythus and newly identified bacteroides phylotype, oral clone BU063, to periodontal health status. The study was accomplished with a set of samples that were collected from subjects with periodontitis and healthy controls. PCR based detection method was used to maximize detection sensitivity. They found the presence of Bacteroides forsythus in the oral cavity was strongly associated with periodontitis, and its nearest genetic neighbour oral clone BU063 was associated with oral health.4 Marlise I Klein, Reginaldo B Goncalves (2003) conducted a study to determine the prevalence of Tannerella forsythia and Porphyromonas gingivalis in subgingival plaque samples using polymerase chain reaction and the reaction of these bacteria was assessed in periodontal health and disease. The results showed that the distribution of Tannerella forsythia was increased in disease sites compared to healthy sites. The study concluded the possible association between periodontal disease and the presence of Tannerella forsythia.5
Hui Wen Yang, Yu Feng Huang, Ming Yung Chou (2004) conducted a study to compare the prevalence & levels of Porphyromonas gingivalis and Tannerella forsythensis in 498 subgingival plaque samples from both healthy individuals and periodontitis patients. Porphyromonas gingivalis & Tannerella forsythia were detected by indirect immunofluorescent assay using species specific polyclonal antisera. The levels of Porphyromonas gingivalis and Tannerella forsythia were found to be increased in diseased subjects compared to healthy subjects.6 6.3 Objectives of the study: The objectives of this study are
To determine the presence of Tannerella forsythia in subgingival plaque samples of periodontally healthy individuals, using polymerase chain reaction and the culture technique.
To determine the presence of Tannerella forsythia in subgingival plaque samples of individuals with chronic periodontitis, using polymerase chain reaction and the culture technique.
To compare the technique of culture and polymerase chain reaction in the identification of Tannerella forsythia in subgingival plaque samples of periodontally healthy individuals and those with chronic periodontitis.
Methodology 7.1 Source of data: It shall be a study to compare the identification of Tannerella forsythia by culture and polymerase chain reaction technique, in periodontally healthy and diseased subjects.
The subjects for the study shall be selected from the patients visiting the Department of Periodontology and the samples shall be analyzed at the Department of Molecular Biology and Immunology at Maratha Mandal’s NGH Institute of Dental Sciences & Research Centre.
7.2 Methods of collection of data: Written informed consent will be taken from all subjects. Plaque index (Silness and Loe) Gingival index (Loe and Silness), Bleeding index (Ainamo and Bay), probing depth and attachment loss will be recorded in all subjects. A total of 50 periodontally healthy subjects and 50 patients diagnosed with chronic periodontitis based on clinical examination will be included for the study.
Subjects will be distributed into 2 groups according to their clinical diagnosis:
i) Periodontally healthy individuals
ii) Chronic Periodontitis group
For Periodontally healthy group : - No signs of gingival inflammation
-Patients with history of any systemic diseases/conditions.
-Pregnant and lactating women
Sample collection: Sampling areas shall be isolated with cotton rolls, supragingivally scaled,and then with the help of a sterile curette, subgingival plaque will be obtained.
Subgingival samples shall be collected from the deepest sites of the periodontal pockets in periodontitis patients and from the gingival sulcus in healthy individuals, and immediately transferred in an appropriate transport medium.
Culture procedures will be carried out in the laboratory of oral microbiology.
Samples will be cultivated under anaerobic conditions on an NAM (N-acetyl-muraminic acid, 10mg/l) agar plate. For polymerase chain reaction analysis, specific primers will be selected. The data obtained will be statistically analyzed using the chi square test. The techniques shall be compared using the sensitivity and the specificity tests.
7.3 Does the study require any investigations to be conducted on patients or
other human or animals? YES
7.4 Has ethical clearance been obtained from your institution in case of 7.3? YES
List of References
Paster BJ et al. Bacterial diversity in human subgingival plaque. J Bacterial 2001;12:3770-783.
Tanner AC, Haffer C, Bratthal GT, Visconti RA, Socransky SS. A study of the bacteria associated with advancing periodontitis in man. J Clin Periodontol 1979;6:278-07.
Anne C R Tanner, Jacques Izard. Tannerella forsythia, a periodontal pathogen entering the genomic era. Periodontol 2000 2006;42:88-113.
Eugene J Leys, Sharon R Lyons, Melvin L Moeschberger, Robert W Rumpf, Ann L Griffen. Association of Bacteroides forsythus and a Novel Bacteroides Phylotype with Periodontitis. J Clin Microbiol 2002;40:3:821-25.
Marlise I.Klein, Reginaldo B Goncalves. Detection of Tannerella forsythensis (Bacteroides forsythus) and Porphyromonas gingivalis by Polymerase Chain Reaction in Subjects with Different Periodontal Status. J Periodontol 2003;74:798-02.
Hui Wen Yang, Yu Feng Huang, Ming Yung Chou. Occurrence of Porphyromonas gingivalis and Tannerella Forsythensis in Periodontally Diseased and Healthy Subjects. J Periodontol 2004;75:1077-83.