Dissertation- Synopsis s1

DISSERTATION- SYNOPSIS

DR. RAKESH RAJEEVAN NAIR

DEPARTMENT OF CONSERVATIVE DENTISTRY AND ENDODONTICS

KVG DENTAL COLLEGE AND HOSPITAL, SULLIA.

BATCH OF 2013-16.

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BANGALORE- KARNATAKA

ANNEXURE- II PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION 1. NAME OF THE CANDIDATE DR. RAKESH RAJEEVAN NAIR AND ADDRESS POST GRADUATE STUDENT DEPARTMENT OF CONSERVATIVE DENTISTRY & ENDODONTICS K.V.G DENTAL COLLEGE & HOSPITAL, KURUNJIBAGH SULLIA- 574327

2. NAME OF THE INSTITUTION K.V.G DENTAL COLLEGE & HOSPITAL SULLIA (D.K), KARNATAKA.INDIA

3. COURSE OF STUDY & SUBJECT MASTER OF DENTAL SURGERY IN CONSERVATIVE DENTISTRY & ENDODONTICS

4. DATE OF ADMISSION TO 27-05-2013 COURSE PCR BASED DETECTION OF THREE 5. TITLE OF THE TOPIC ANAEROBIC BACTERIA ASSOCIATED WITH ENDODONTIC-PERIODONTIC LESIONS IN NON-DIABETIC AND TYPE- 2 DIABETIC SUBJECTS 6. BRIEF RESUME OF THE INTENDED WORK

6.1 Need of the study

The endodontic and periodontal systems are closely related and the disease occurrence can overlap into each other.1 Lateral and accessory canals can transport toxic substances and facilitate the bacterial colonization of dental tissues. These toxins and microbial agents can induce periodontal inflammation.2 Studies show that pulpal and periodontal diseases are responsible for more than 50% of tooth mortality. 3 Moreover the differential diagnosis of endo-periodontal disease is difficult since they are caused by microbes which are typically poly microbial in nature.4

A reciprocal relationship exists between glycemic control and chronic periapical lesions. Treating infections of pulp and periodontium will improve glycemic control and help in healing of lesions similar to non-diabetics.5There is an increase of tissue destruction in diabetic periodontitis, and periodontal infection may complicate the severity of diabetes and the degree of metabolic control, resulting in a 2- way relationship between diabetes mellitus and periodontal disease.6 The number of people with diabetes in India is around 40.9 million and is expected to rise to 69.9 million by 2025 according to the statistics by international diabetes federation in 2007.7

PCR is the most sensitive of the existing methods to detect microbial pathogens that are difficult to culture and it has the advantages of being more sensitive, accurate and rapid compared to other methods. Identification of bacteria associated with endodontic and periodontal lesions indicates both antagonistic & synergistic interactions between different strains & species.4

The diabetic host may have an increased periapical lesion size or may develop more serious infections in response to virulent root canal bacteria.8 Assessment of microbes in teeth with endodontic - periodontal lesions especially in these immune compromised patients offer new perspectives for diagnosis, targeted treatment and subsequently for the better prognosis of the affected teeth .1 To date literature on identification of bacterial species isolated from endodontic-periodontal lesions in diabetics is sparse. Hence the study. 6.2 Review of Literature:

A PCR based study for the identification of bacteria associated with endodontic infections was conducted. PCR primers that targeted the bacterial 16S rRNA genes were used to identify ten putative pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were investigated. Microbial samples from the root canals of 24 teeth with necrotic pulp were included of which six were diabetic. The access cavity was prepared with a new sterile endo z bur. Three fine paper points were left in the canal for 30 seconds and sampling was done. The paper points were placed in DNA and RNA free vials. The vials were vortexed for 2 minutes to disperse microbial cellular materials into suspension sterile 1mM EDTA.PCR evaluation was done using universal bacterial as well as species or genus specific PCR primers. Eight specimens were positive for Enterococcus spp. Of these, 6 (19%) were from non-diabetic and 2 (33%) from diabetic patients. PCR with specific primers showed that preoperative symptoms were significantly associated with the presence of Streptococcus spp. There was also a nonsignificant trend for symptoms to be associated with Fusobacterium nucleatum and Porphyromonas gingivalis and for diabetes mellitus to be associated with Porphyromonas gingivalis and Porphyromonas endodontalis. Cloning and sequencing of the universal PCR product in one specimen revealed the presence of an organism related to the genus Olsenella, which has not previously been described in endodontic infections. Bacterial combinations in root canals may be more pathogenic than individual strains. They concluded that it is important to determine the association of bacterial combinations with clinical signs and symptoms or treatment outcome, as well as the association of certain microorganisms with each other 8

Investigation of five periopathogens: Aggregatibacter actinomycetemcomitan, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia in patients with endo- periodontal lesions was done using PCR. Forty-one patients, 26 of them with necrotic pulp, and 15 with incomplete root canal fillings, combined with periodontal lesions, were investigated. Microbial investigation of the endodontic and periodontic biofilms and semi qualitative assessment of bacteria was performed with genetic assay. A clinical examination was performed, together with periapical radiographs and microbiological investigation of the endodontic and periodontal biofilms. Clinical examination included probing depth, plaque index, tooth mobility, furcation involvement, and presence of untreated necrotic pulp presence/absence of apical lesion, sensitive to percussion. Periodontal and root canal samples were taken with help of paper points kept for one minute stored, frozen at -80o in sterile 2ml cryo tube of PBS. Qualitative and semi-quantitative evaluation of the proposed bacteria was performed by a molecular genetic assay based on multiplex amplification with biotinylated primers and reverse hybridization. Results showed the most prevalent species in endodontic samples were Tannerella forsythia (65.8%), Porphyromonas gingivalis (61%).Periodontal samples were frequently positive for Treponema denticola 80%, Porphyromonas gingivalis 5%, Tannerella forsythia 78 %, Prevotealla intermedia(73%) . Tannerella forsythia and Prevotealla intermedia had a positive association in endodontic samples as well as Tannerella forsythia and Porphyromonas gingivalis in periodontal samples. They suggested that endodontic infections might constitute a reservoir of periodontal pathogens. Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia of the five investigated species may play specific roles in the pathogenesis of endodontic-periodontal lesions.9

Investigation of six selected bacterial species in endo-periodontal lesions was done using PCR.Association of six tested bacteria belonging to orange and green complex:Parvimonas micra, Fusobacterium nucleatum, Campylobacter rectus, Eubacterium nodatum, Eikenella corrodens and Capnocytophaga sputigena was evaluated. Forty-six patients presenting with endodontic-periodontal lesions in root canal treated teeth and teeth without previous root canal treatment were evaluated. The widest and the most permeable root canal adjacent to the periodontal lesion was then sampled. Rotary pro-taper universal (Dentsply Maillefer, Switzerland) nickel-titanium instruments were used in a crown down motion at 500rpm along with a torque controlled engine(Endomate TC, NSK, Japan).Clinical examinations, periapical radiographs and microbiological sampling from the canal system and periodontal pockets were performed using PCR and DNA-DNA hybridization. Extremely high bacterial loads in endodontic samples were recorded for P. micra, F. nucleatum and C. sputigena, while periodontal samples were often colonized by the same species, plus C. rectus. Significant association was recorded between F. nucleatum-endo and P. micra-endo. The findings suggest that F. nucleatum, P. micra and C. sputigena may play a role in the pathogenesis of endodontic-periodontal lesions.4

Detection and clonal analysis of anaerobic bacteria associated to endodontic periodontic lesions was evaluated. Twenty-seven teeth of patients with endodontic-periodontal lesions were selected. Samples were spread on an agar-blood medium, the detection of each species was performed using a polymerase chain reaction. The determination of the simultaneous presence of the same species in the micro environment by one or more clones was determined by using arbitrarily primed PCR. The results showed Prevotella intermedia was the most prevalent species of the colonies in periodontal pockets, whereas Porphyromonas gingivalis and Prevotella intermedia were the more prevalent in root canals. Isolates of Prevotella intermedia and Porphyromonas gingivalis were simultaneously identified in root canals and periodontal pockets. Eighteen percent of teeth exhibited the simultaneous colonization by Pg, Tannerella forsythia (previously T. forsythensis), and Porphyromonas endodontalis in the pulp and periodontal microenvironments. They concluded that different clones of prevotella intermedia and porphyromonas gingivalis with a high intraspecific genotype similarity were found to colonize the same anatomic sites in endodontic- periondontal infections.2

The present review article analyzed microbial aspects of endoperiodontal lesions by a search strategy. Comprising the electronic studies of data basis such as PubMed, Science direct, and Cochrane etc. The results showed that there were similarities in the endodontic and periodontal micro flora. However, the number of microorganisms within the cross infection is limited, including Bacteroids, Eubacteria, Spirochetes and Wolinella. The bacteria forming the red complex are closely related to the severity of the periodontal disease and can participate in the pathogenesis of the periradicular abscess.They concluded that there are many communication routes between the periodontium and pulpal tissue. Therefore contamination from one tissue to another can occur, existing a microbiological interrelationship between these tissues1

The article reviews the role of diabetic mellitus from an endodontic perspective. A detailed assessment of the literature on diabetes mellitus and its implication of pulp and peri apical disease related to the prevalence of periapical lesions in diabetics, pathogenesis of periapical lesions in diabetics, effect of diabetes in endodontic treatment outcome were discussed. Studies indicate a higher prevalence of apicalperiodontitis in diabetics (81.3%) compared with non-diabetics (58%).They suggested that diabetes may serve as a disease modifier of periapical lesions. A reciprocal relationship exists between glycaemiccontrol and chronic periapical lesions. Treating infections of pulp and periodontium will

improve glycemic control and help in healing of lesions similar to non-diabetics. To provide competent care to patients with DM, dental clinicians must understand the disease, its treatment, and its impact on the patient`s ability to undergo and respond to endodontic treatment. DM affects people of all age groups, and its prevalence has been increasing. Therefore more and more diabetic patients will be requiring endodontic treatment. Research indicates increased prevalence of periapical lesions in diabetics, with decreased success rate of endodontic treatment. A reciprocal relationship exists between glycemic control and chronic periapical lesions. Hence it is very vital for dental clinicians to understand the disease process of DM, its effect on pulp and periapical diseases, and their treatment outcome to provide competent endodontic treatment to patients with Diabetic mellitus.5

6.3 Aim and objective of the Study:

To clinically isolate and detect three anaerobic bacteria associated with endo-periodontal lesions in non- diabetic and type -2 diabetic subjects using PCR.

Objectives:

a) To isolate and detect the association of Porphyromonas gingivalis,Prevotella intermedia and Enterococcus faecalis in endo-periodontal lesions in non-diabetic subjects using PCR

b) To isolate and detect the association of Porphyromonas gingivalis,Prevotella intermedia and Enterococcus faecalis in endo-periodontal lesions in type-2 diabetic subjects using PCR.

c) To compare and analyze the above two groups.

d) To evaluate the association of Porphyromonas gingivalis,Prevotella intermedia and Enterococcus faecalis and relate the signs and symptoms with these microorganisms associated with endo-periodontal lesions of non-diabetic and type 2 diabetic subjects.

7 MATERIAL AND METHODS:

7.1 Source of the Data: 60 patients with endodontic-periodontal lesions in which 30 are non-diabetic and 30 are type 2 diabetic subjects will be selected with age group of 30-60 years referred for endodontic-periodontal treatment to KVG Dental College and Hospital, Sullia, Karnataka.

7.2 Method of Collection of Data

-All the 60 subjects(30 non-diabetic and 30 type -2 diabetic) of age group 30-60 years requiring endodontic-periodontic treatment will be randomly selected and divided into two groups of 30 teeth in each group.

Group 1 (30 No`s) - Healthy Non diabetic subjects with endodontic-periodontic lesions.

Group 2 (30 No’s) – Type-2 diabetic subjects with endodontic -periodontic lesions.

WHO criteria for diabetes mellitus - RBS > 200 mg/dl or FBS > 126mg/dl, or 2hr PPBS > 200 mg/dl or glycosylated HB > 6.5% will be assessed.

Informed Consent and Ethical Clearance:

The study will be initiated subsequent to approval of ethical committee. Consent of patients willing to participate in the study obtained in a given format (attached document no: 1). A detailed medical and dental history will be obtained from each patient.

Inclusion Criteria

1. Patients aged 30-60 years

2. Both males and females will be included.

3. Patient requiring treatment for endodontic-periodontal lesions and those willing to participate in the study. 4. For the diagnosis of endodontic-periodontal lesions the following clinical-radiographic factors are considered: periodontal probing depth >5mm, , Boneloss, Clinical attachment loss, Presence of inflammation, Bleeding on probing, Irreversible pulpitis with chronic apical periodontitis

5. The subjects in group 2 will have a random blood sugar level > 200mg/dl, Fasting blood sugar level >126mg/dl, 2hr Post prandial blood sugar level > 200mg/dl and glycosylated HB > 6.5%.

Exclusion Criteria:

1. Any systemic diseases for group 1 and systemic diseases other than type-2 diabetes for

Group 2.

2. Pregnancy and lactation

3. Teeth with root fracture

4. Use of any antibiotics in past 3 months

5. Teeth that cannot be isolated with rubber dam

6. Tortuous canals

7. Teeth with developmental defects.

Data collection and Sampling size:

Clinical samples from 60 root canals and periodontal pockets of 30 non diabetic and 30 diabetic subjects of the age group of 30-60 years diagnosed as endodontic-periodontic lesion in any teeth will be selected.

CLINICAL EXAMINATION

Case history and clinical examination of all the subjects will be evaluated and recorded as follows. CLINICAL & RADIOLOGICAL EXAMINATION

Periodon Clinical Mobi Furcation Sinus Pain Swelling Tender Tooth Caries Wideni Periapica Bone tal attachment lity involvement tract on Vitality ng of l lesion loss Probing loss(mm) percussi Testing periodo size depth on ntal ligamen (mm) t space

Radiographic Examination:

Each tooth will be evaluated for the presence of a radiographically detectible pathosis at time of diagnosis and treatment. The size of the lesion was calculated by taking the average of the lesion’s largest dimension and the width perpendicular to the largest dimension.

Pre-operative assessment of periapical status:

Strindberg’s and Orstavik’s criteria was followed.

Criteria by Orstavik et al. (1986)

For all the teeth, the presence of a periapical radiolucency is assessed using the periapical index (PAI), determined with a paralleling X-ray technique according to Orstavik et al. (1986). Teeth with a PAI score equal to or greater than 3 were considered to be affected by (peri) apical bone lesions.

Each of the roots was categorized as:Scores:

1- Normal periapical structure

2- Small changes in bone structure

3- Changes in bone structure with some mineral loss 4- Periodontitis with well-defined radiolucent area

5- Severe periodontitis with exacerbating features

Strindberg’s (1956) criteria

The contour and width of the periodontal ligament:

1 < 3mm 2 3-5mm 3 >5mm

CLASSIFICATION OF ENDO PERIO LESIONS ACCORDING TO BELK & GUTMANN (1990)

 Type-1.Primary endodontic lesions with secondary periodontal involvement

 Type-2.Primary periodontal lesions with secondary endodontic involvement

 Type-3.True combined endodontic-periodontal lesions or concomitant pulpal and periodontal lesions

GLICKMAN CLASSIFICATION (FURCATION INVOLVEMENT)

Class I

 Incipient or early stage of furcation involvement.

Class II

 Cul-de-sac involvement

 Vertical bone loss may be present

Class III  Through and through involvement

Class IV

 Through and through with no soft tissue occluding the openings

 Complete visualization possible9

PROBING DEPTH

The periodontal probe (PCP 15 – HuFriedy, Chicago, IL, USA) will be inserted parallel to the vertical axis of the tooth and `walked’ circumferentially clockwise around each surface of the tooth, to detect the area of deepest penetration. A probing depth >5mm is considered as a periodontal pocket. CLINICAL ATTACHMENT LOSS (Carranza)

The distance from the cemento enamel junction to the base of the pocket represents the clinical attachment loss will be assessed.

TOOTH MOBILITY (Miller`s classification)

Class 0

 Normal (physiologic) movement when force is applied.

Class I

 Mobility greater than physiologic.

Class II

 Tooth can be moved up to 1mm or more in a lateral direction (buccolingual or mesiodistal).  Inability to depress the tooth in a vertical direction (apicocoronal).

Class III

 Tooth can be moved 1mm or more in a lateral direction (buccolingual or mesiodistal).  Ability to depress the tooth in a vertical direction (apicocoronal).

Assessment of periradicular infections

Acute apical periodontitis, chronic apical periodontitis and sub-acute apical periodontitis, external root resorption, widening of the periodontal ligament space as assessed by signs and symptoms and radiological findings.

Armamentarium and Materials

1. Diagnostic Instruments: Mouth Mirror, Explorer, and Tweezers.

2. Electrical pulp tester

3. X-rays

4. Lignocaine with Adrenaline 5. Disposable Syringes

6. Rubber dam

7. Spoon Excavator

8. Airotor handpiece

9. Micromotor handpiece

10. Anthogyr handpiece

11. Access cavity burs.(Endo Z carbide bur)

12. Ultra sonic scaler

13. Pumice

14. 30% hydrogen peroxide

15. 2.5% sodium hypochlorite solution

16. 5% sodium thiosulphate

17. #15 K-type file

18. Paper points

19. 0.9% Sterile Saline solution

20. Eppendorf tubes

21. Test tubes

22. RTF solution

23. PCR machine

24. Eppendorf Master cycler

25. Eppendorf Centrifuge 5415-D

26. Eppendorf Pipette 27. Eppendorf Pipette tips

28. Bacterial primers.

Porhyromonas gingivalis - AGG CAG CTT GCC ATA CTG CG

ACT GTT AGC AAC TAC CGA TGT

Prevotella intermedia - CGT GGA CCA AAG ATT CAT CGGTGG A CCG CTT TAC TCC CCA ACA AA Enterococcus Spp. - TAC TGA CAA ACC ATT CAT GAT G

AAC TTC GTC ACC AAC GCG AAC

ISOLATION PROCEDURE AND TOOTH DECONTAMINATION:

All the treatment and sampling procedures will be done by the same endodontic specialist and will be collected under strictly aseptic conditions. Each tooth will be cleansed with pumice and isolated with rubber dam. The tooth and surrounding field will be then cleansed with 30% hydrogen peroxide (H202) and decontaminated with 2.5% of sodium hypochlorite solution (NaOCL).

ENDODONTIC ACCESS:

It will be achieved with a sterile high speed Endo Z carbide bur. After access is achieved the tooth and the adjacent rubber dam will be once again disinfected with 30% H202 and 2.5% NaOCL. The cavity will be swabbed with 55% sodium thiosulfate solution to inactivate the NaOCL

EVALUATING THE EFFICACY OF THE ISOLATION PROCEDURE:

Sequential sterile cotton pellets will be moistened in sterile saline solution (0.9% NaCl) to swab the access cavity and the tooth surface. This will be then transferred to a vial containing 0.75 mL of RTF solution and sampled for bacterial growth quality control [QC]. If growth occurs, the patient sample will be disqualified from the study.

SPECIMEN SAMPLING

Sample collection from periodontal pockets. The supra gingival plaque will be removed with an ultrasonic device and with gentle rotary brushing. The periodontal sites to be sampled will be air dried and isolated with cotton rolls and two sterile paper points ISO size 40 will be inserted into periodontal pocket, after one minute, the paper points will be removed & placed in RTF solution and incubated and will be microbiologically assessed by PCR

Sample collection from endodontic site.

The tooth of interest is isolated with rubber dam. After cleaning and decontamination of the tooth and surrounding tissues (1 mL of 3% hydrogen peroxide for cleaning, and 1 mL of 2.5% NaOCl solution for antisepsis).Access preparation is done with sterile burs without water spray. In case of multi rooted teeth, roots with apical lesions were taken into consideration for sampling; if more than one root per tooth had an apical lesion, the widest and most permeable root canal adjacent to the lesion is then sampled and working length will be established. Universal titanium instruments will be used at 500 rpm in a crown – down motion with a torque controlled engine according to manufacturer’s instructions. Samples are initially collected by means of a size 15 k-file. The file will be introduced approximately 1 mm short of the foramen, and a discrete filing motion is applied to obtain the sample. Later two size 20 paper points will be placed at the same level to soak up the fluid in the canal. Then the paper points are transferred into RTF solution and will be immediately incubated.1

All the samples will be transported to the microbiology department of St: Aloysius College, Mangalore for PCR evaluation.

PCR assay

DNA extraction

The root canal samples in the transport medium will be thawed to 37°C for 10 minutes and homogenized by vortex-mixing for 1 minute. The paper points are removed, and the microbial suspension is washed 3 times with 200µl of ultrapure water by centrifugation for 2 minutes at 2500g. After the final wash, the pellets are resuspended in 200µl of ultrapure water, boiled for 10minutes in a water bath, quickly chilled by placing on ice for 5 minutes, and centrifuged at 9000g at 4°C to remove unbroken cells and large debris. The supernatant is then collected and used as the template for PCR amplification.

Reference DNA from several species will be extracted to serve as positive control for the taxon-specific primers used or to evaluate the specificity of the primers. PCR Amplification

The PCR reaction used to assess the occurrence of all target taxa, is performed in 50 μl of reaction mixture containing 38 μl sterile distilled water,5 μl 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl

(pH 9.0), 1.0% Triton X 100),3 μl 25 mM MgCl2, 1 μl 10 mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP), 1 μl 20 μM forward primer,1 μl 20 μl reverse primer, 0.2-1 μl Taq polymerase. Negative controls consisting of ultrapure water instead of sample will be included with each batch of samples analyzed.

DNA amplification is performed in a thermal cycler system (Eppendorf Master Cycler). Amplicons are stored at –20oC. The amplification products will be analyzed through the use of electrophoresis in a 1.5% agarose gel conducted at 4V/cm in Tris-borate EDTA buffer.

Gel electrophoresis

Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA. 1% (w/v) Agarose gel is prepared by mixing 1g of Agarose with 100mls of 1X TBE buffer in a 250ml flask. This mixture is then heated carefully in a microwave up to its boiling point with occasional gentle swirling. The agarose gel is left to cool before 2.5ul of 10mg/ml ethidium bromide solution per 100ml gel was added and mixed gently. This allows the DNA to be visualized under UV light. The ends of the gel former are sealed with tape and the agarose gel is poured into it and left to set. When the gel is set, tape is removed and placed into an electrophoresis tank and 1X TBE buffer is poured into the tank until it is about 1m above the gel. 5ul of the loading dye is added to each of the eppendorf tubes. This aids the loading into the wells by increasing the sample density and allows us to see how far the samples migrate. 5ul of 100BP DNA Ladder is loaded into a well and 20µl of each of the PCR samples are loaded into the wells beside it. The voltage on the tank is then turned on so that electrophoresis occurs. The electrophoresis is stopped before the dye runs of the end of the gel. Gels are then viewed on a UV transilluminator under 300 nm ultraviolet light and a photographic image is captured to be analyzed. The identity of each band will be determined by visual comparison with a molecular weight ladder of the three microorganisms to be tested. Reactions are deemed positive in the presence of bands of the appropriate size. Results will be recorded on Spreadsheet for analysis.

Follow up: Nil

Follow up period: Nil

Analysis of data

Analysis will be done using Chi – square test, Mann – Whitney or Odds ratio. Flow chart of investigation design

60 subjects (30 non diabetic and 30 type 2 diabetic subjects) with endodontic periodontal lesions referred to KVG Dental College and Hospital, Sullia will be included in the study.

Informed consent from all patients will be taken

Charting of records - medical and dental records including signs and symptoms.

History Clinical features Radiographic features

Root Canal access opening & working length determination

Sampling from endodontic site Sampling from periodontal site

Isolation and detection of Porphyromonas gingivalis, Prevotella intermedia, and Enterococcus faecalis using species specific PCR at St:Aloysius College,Mangalore.

Data analysis will be done using Chi-square test, Mann-Whitney test & Odds ratio

Completion of root canal treatment 7.3 Does the study require any investigation\intervention to be conducted on patients\ humans\ animals? If so, please describe briefly:

Yes. Diabetic screening will be done and radiographic investigation will be carried out for all the 60 subjects. The study does not inflict any harm to the patients .This study will be done under the supervision of my guide and co-guide.

7.4 Has ethical clearance been obtained from your institution in case of 7.3?

Yes. Copy of ethical clearance certificate has been attached. 8. REFERENCES 1. Tokunaga C, Tomazinho FSF. Microbiological aspects of endoperiodontal lesion. Rev. Sul-bras. Odonto. 2013;10(2):176-81. 2. Pereira CV, Stipp RN, Fonseca DC, Pereira LJ, Hofling JF. Detection and clonal analysis of anaerobic bacteria associated to endodontic–periodontic lesions. J Periodontol 2011;82(12):1767-75. 3. Dr.Peeran SW, Thiruneervannan M, Abdalla KA, Mugrabi MH. Endo-perio lesions. Int J Sci Tech Res, 2013;2(5):268-74. 4. Didilescue AC, Rusu D, Anghel A, Nica L, Iliescu A, Greabu M, et al.Investigation of six selected bacterial species in endo-periodontal lesions. Int Endod J. 2012;45(3):282-93. 5. Chakravarthy PVK. Diabetes mellitus: an endodontic perspective. European J Gen Dent, 2013;2:241-45. 6. Grossi SG, Genco RJ. Periodontal disease and diabetes Mellitus: a two-way relationship. J Periodontol, 1998;3(1):51-61. 7. Mohan V, Sandeep S, Deepa R, Shah B, Varghese C. Epidemiology of type 2 diabetes: Indian scenario. Indian J Med Res. 2007 Mar;125(3):217-30. 8. Foud AF, Barry J, Caimano M, Clawson M, Zhu Q, Carver R, et al. PCR-based identification of bacteria associated with endodontic infections. J Clin Microbiol. 2002;40(9):3223–31. 9. Didilescu AC, Rusu D, Greabu M, Iliescu AA, Bancescu G, Anghel A, et al. Investigation of five periopathogens in patients with endo-periodontal lesions.Journal de Parodontologie & d’Implantologie Orale.2010;30(2). 10. Nair PNR. Pathogenesis of apical periodontitis and the causes of endodontic failures. Crit Rev Oral Biol Med.2004;15(6):348-381. 11. Baumgartner JC, Siqueira JF Jr, Xia T, Róças IN. Geographical differences in bacteria detected in endodontic infections using polymerase chain reaction. J Endod 2004;30(3):141-144. 12. Sunitha RV, Emmadi P, Namasivayam A, Thyegarajan R,Rajaraman V. The periodontal – endodontic continuum:a review. J Conserv Dent. 2008;11(2):54–62. 13. Nair PN. Apical periodontitis: a dynamic encounter between root canal infection and host response. Periodontol 2000. 1997;13:121-48. 14. Yamamoto Y.PCR in diagnosis of infection: detection of bacteria in cerebrospinal fluids. Clin Diagn Lab Immunol. 2002;9(3):508–514. 9. SIGNATURE OF CANDIDATE 10. REMARKS OF THE GUIDE 11. NAME AND DESIGNATION OF (IN BLOCK LETTERS) 11.1 Guide Dr. MOKSHA NAYAK. M.D.S. PRINCIPAL AND PROFESSOR , DEPARTMENT OF CONSERVATIVE DENTISTRY AND ENDODONTICS, K.V.G DENTAL COLLEGE AND HOSPITAL KURUNJIBAG, SULLIA D.K -574327 11.2 Signature

11.3 Co-Guide: (IF ANY) Dr. SUBBANNAYYA KOTIGADDE. Ph.D PROFESSOR, DEPARTMENT OF MICROBIOLOGY, K.V.G MEDICAL COLLEGE AND HOSPITAL KURUNJIBAG, SULLIA D.K-574327 11.4 Signature 11.5 Head Of The Department Dr. KRISHNAPRASADA L. MDS, DNB, MBA. HOD AND PROFESSOR, DEPARTMENT OF CONSERVATIVE DENTISTRY AND ENDODONTICS, K.V.G DENTAL COLLEGE AND HOSPITAL KURUNJIBAG, SULLIA D.K -574327 11.6 Signature 12. 12.1 Remarks Of The Principal

12.2 Signature And Official Seal

Document no: 1.

K. V. G. DENTAL COLLEGE & HOSPITAL

KURUNJIBAG - 574 327, SULLlA, D. K., KARNATAKA, INDIA

SPONSORED BY ACADEMY OF LIBERAL EDUCATION (REGD.) SULLIA

Department of Conservative Dentistry and Endodontics

INFORMED CONSENT FOR PARTICIPATION IN RESEARCH

I, Dr. RAKESH RAJEEVAN NAIR, PG, Department of Conservative Dentistry And Endodontics,I am conducting a research work for Award of MDS degree in Conservative Dentistry and Endodontics.

The topic for the study is: PCR BASED DETECTION OF THREE ANAEROBIC BACTERIA ASSOCIATED WITH ENDODONTIC - PERIODONTIC LESIONS IN NON- DIABETIC AND TYPE-2 DIABETIC PATIENTS.

Objectives:

a) To isolate and detect the association of Porphyromonas gingivalis, Prevotella intermedia and Enterococcus faecalis in endo periodontal lesions in non-diabetic subjects using PCR

b) To isolate and detect the association of Porphyromonas gingivalis, Prevotella intermedia and Enterococcus faecalis in endo periodontal lesions in type -2 diabetic subjects using PCR.

c) To compare and analyze the above two groups.

d) To evaluate the association of Porphyromonas gingivalis, Prevotella intermedia and Enterococcus faecalis with signs and symptoms and history of type-2 diabetes mellitus using PCR. I ______have been told in my own language that I understand about the study. I have been told that this is for a research procedure, that my participation is voluntary and I/he/she reserve the full right to withdraw from the study at my own initiative at any time, without having to give any reason, and that right to participate or with draw from the study at any stage will not prejudice my/his/her, rights and welfare. Confidentiality will be maintained and only be shared for academic purposes.

Privacy and Confidentiality The only people to know that you are a research subject are members of the research team. No information about you or provided by you during the research will be disclosed to others without your written permission except 1. In emergency to protect your rights and welfare. 2. If required by law

Financial incentives for participation You will not be paid/offered any free gifts for participating in the research.

I here by give consent to participate in the above study. I am also aware that I can withdraw this consent at any later date, if I wish to. This consent form being signed voluntarily indicating my agreement to participate in the study, until I decide otherwise. I understood that I will receive a signed and dated copy of this form. I have signed this consent form, before my participation in this study. If I have any question/doubt pertaining to the study, I have been asked to contact Dr.Rakesh Rajeevan Nair , mobile no- 8105750521

Signature of the research subject: Date: Place: Signature of the witness: Date: Place: I here by state that the study procedures in details were explained and all questions were fully and clearly answered to the above mentioned participant /his/her relative

Investigators signature: Date: Place: Investigator Certificate

I certify that all the elements including the nature, purpose and possible risks of the above study as described in this consent document have been fully explained to the subject. In my judgment, the participant possesses the legal capacity to give informed consent to participate in this research and is voluntarily and knowingly giving informed consent to participate.

Signature of the Investigator: ______Dated:______Name of the Investigator: ______