DISSERTATION- SYNOPSIS

DR.GRISHMA FLORENCE NORONHA

DEPARTMENT OF CONSERVATIVE DENTISTRY

AND ENDODONTICS

KVG DENTAL COLLEGE AND HOSPITAL,

SULLIA.

BATCH OF 2011-14.

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA,

BANGALORE

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. NAME OF THE CANDIDATES

AND ADDRESS

(IN BLOCK LETTERS)

DR GRISHMA FLORENCE NORONHA

DEPARTMENT OF CONSERVATIVE

DENTISTRY AND ENDODONTICS

KVG DENTAL COLLEGE AND HOSPITAL,

KURUNJIBAG

SULLIA, KARNATAKA.

2. NAME OF THE INSTITUTION KVG DENTAL COLLEGE AND HOSPITAL

SULLIA

3. COURSE OF STUDY AND

SUBJECT

MASTERS IN CONSERVATIVE

DENTISTRY AND ENDODONTICS

4. DATE OF ADMISSION TO

COURSE

26-05-2011

5. TITLE OF THE TOPIC: DETECTION AND IDENTIFICATION OF

ENTEROCOCCUS FAECALIS ,

STREPTOCOCCUS SPECIES AND

CANDIDA ALBICANS IN SECONDARY

ROOT CANAL INFECTION OF NON

DIABETIC AND TYPE-2 DIABETIC

PATIENTS : SPECTROSCOPIC

SIGNATURE DEVELOPMENT AND

EVALUATION.

6. BRIEF RESUME OF THE INTENDED WORK:

6.1 Need for the study

Persistent intraradicular or secondary infection seem to be the major cause of root canal

treatment failure, which is usually characterized by radiographic evidence of periapical

lesions.1 Studies have shown endodontic failure rate to be 16%, which means 84% success rate.2

PCR analysis of microbes from filled root canals with persistent periapical disease have

recovered 77% Enterococcus species, 23% Streptococcus species and 9% Candida species.3

Diabetes mellitus (DM) is a common metabolic disorder of carbohydrate and lipid

metabolism.4 India leads the world with largest number of diabetic subjects earning the dubious

distinction of being termed the “diabetes capital of the world”.5 The International Diabetes

Federation (IDF) estimates the total number of people in India with diabetes rising to 87.0

million by 2030.6 Clinical and radiographic studies by the investigators have shown a greater

prevalence of periapical lesions in diabetics than in nondiabetics.4

Currently, there are a variety of microbiology, immunoassay, genetic, and molecular-

based approaches for the identification of endodontic microorganisms. However, traditional

methods are limited by long analysis times associated with culture sample collection and

preparation, DNA extraction, and reliance on reagents with limited shelf lives and also require

highly trained personnel to identify microorganisms contained within a sample.7,8 Biochemical

identification of bacteria after isolation in pure culture is necessary, even if it is labor-intensive

and time-consuming. Identification with a single method such as PCR and 16srRNA gene

sequencing could lead to false-positive results for some bacteria such as oral streptococci ,

leading to the need for a combination of methods to confirm their biochemical identification.9

Spectroscopy has the potential to satisfy the need for rapid detection of threat agents, within

complex matrixes and without the use of costly consumables.7 Laser raman spectroscopy has

shown as a potential chair-side microbiological diagnostic device for isolates obtained from

American type culture collection which have been implicated in various endodontic, periodontic

and other medically important infections.8 However no clinical relevant data is available on

estimation of endodontic microorganisms using Spectroscopy. This thesis presents

spectroscopy-based detection, identification and confirmation of few frequently occurring

endodontic microorganisms of retreatment cases in non diabetic and diabetic subjects.

6.2 Review of Literature:

Factors associated with endodontic treatment failures were assessed. Two hundred and thirty

six cases, none of which had advanced periodontal disease, postperforations, or root or crown

fractures were analyzed clinically, radiographically, and histobacteriologically to determine the

major factor(s) for treatment failures. The samples were obtained from biopsies during

endodontic surgery and serial sections of 6 µm were cut and stained with hematoxylin and eosin

and modified Brown and Brenn. The radiographic diagnosis of preoperative periradicular status,

the radiographic apical extent of root canal fillings, and the histobacteriological findings of

biopsied samples were recorded and correlated. The collected data were subjected to statistical

analysis using the chi-square test. It was found that there was a correlation between bacterial

infection in the canal system and the presence of periradicular rarefaction in endodontic failures.

This report provides evidence indicating that the major factors associated with endodontic

failures were the persistence of bacterial infection in the canal space and/or the periradicular

area and the presence of preoperative periradicular rarefaction. The apical extent of root canal

fillings, had no correlation to treatment failures.10

Microbiological status of 100 root-filled teeth with apical periodontitis and 20 non pathological

teeth were evaluated. The samples were cultured on one Brucella agar plate for incubation in

air with 10% carbon dioxide for 3-5days and on another for anerobic incubation, using the

hydrogen cumbustion method for 5-7 days.117 strains of bacteria were recovered in 68 apical

periodontitis case. Facultative anaerobic species were predominated among these isolates (69%

of identified strains). Growth was classified as ‘sparse’ or ‘very sparse’ in 53%, and as ‘heavy’

or ‘very heavy’ in 42%. Enterococci were the most frequently isolated genera, showing ‘heavy’

or ‘very heavy’ growth in 25 out of 32 cases (78%). No bacteria were recovered from 11 non

pathological teeth whilst the remaining nine yielded 13 microbial strains. It was concluded that

the microflora of the obturated canal differs from that found normally in the untreated necrotic

dental pulp, quantitatively as well as qualitatively.11

The effect of Diabetes mellitus on endodontic treatment outcome was investigated using a

custom built electronic record system in patients with and without diabetes. The medical history

and treatment data for non surgical endodontic patients treated in speciality clinics were entered

in electronic record system. A total of 5,499 (284 with diabetes) non surgical endodontic

patients and 540 (73 with diabetes) follow up patients were recorded . Results showed patients

with diabetes had increased periodontal disease in teeth involved endodontically and have a

reduced likelihood of success of endodontic treatment in cases with preoperative periradicular

lesions.12

The prevalence of apical periodontitis in patients with and without type 2 diabetes was studied.

The records of 38 subjects with diabetes and 32 control subjects were examined. All

participants underwent a full-mouth radiographic survey incorporating 14 periapical

radiographs. Periapical status was assessed using the periapical index score. Apical periodontitis

was found in atleast one tooth in 81.3% of diabetic patients & in 58% of control subjects. They

concluded that type 2 diabetes mellitus is significantly associated with an increased prevalence

of apical periodontitis.13

Raman chemical imaging spectroscopy, reagentless detection and identification of pathogens

using signature development and evaluation was reported. The rationale and steps for

constructing a pathogen Raman signature library were described, as well as the first reported

Raman spectra from live, priority pathogens, including Bacillus anthracis, Yersinia pestis,

Burkholderia mallei, Francisella tularensis, Brucella abortus, and ricin. Most strains were

obtained from the American Type Culture Collection while others came from established

government collections. Presumptive pathogen particles exhibiting properties consistent with

pathogen morphology and fluorescence signatures were analyzed using RCIS. Principal

component analysis (PCA) was employed as a data reduction step. The vector normalized

raman spectral intensisties between 700 and 3200 cm-1 provided the discrimination variables for

PCA. The identity of an agent was evaluated by calculating the mahalanobis distance between a

test spectrum and classes defined is based on the covariance of standard deviation values of the

spectral points in PC data space. Results from a government-managed blind trial evaluation of

the signature library demonstrated excellent specificity under controlled laboratory conditions.7

This study aimed to determine if Laser raman spectroscopy can discern the uniqueness among

10 different species of bacteria contained within a medium in unprocessed and processed

samples. The bacterial species were obtained from American type culture collection which have

been implicated in various endodontic, periodontal, or other medically important infections.

Bacteria were grown in blood agar plate for 3 days .Checkerboard DNA-DNA hybridization

was used for species verification. For unprocessed samples the undisturbed bacterial growth

was transferred to Barium flouride slide and laser scanned. For processed sample the, the

bacterial cells were harvested, washed and resuspended in phosphate buffer saline at 109

cells/ml concentration and then transferred to BaFl2 slide and laser scanned. Selected regions of

raman spectra for each species were processed using two sided t test. Results showed that each

bacterial species generated restricted ranges of unique spectral signatures that were not masked

by the containing media. Chair side LRS is a promising technique that differentiates among oral

bacterial species with a high degree of specificity.8

6.3 Objectives of the Study:

Aim-.Detection and Identification of Enterococcus faecalis, Streptococcus species and Candida

albicans in secondary root canal infection of non diabetic and type-2 diabetic patients using

spectroscopic signature development and evaluation.

Objectives-

A. To investigate Enterococcus faecalis, Streptococcus Species and Candida Albicans in root -

filled teeth with apical periodontits of non diabetic patients using spectroscopy.

B. To investigate Enterococcus faecalis , Streptococcus Species and Candida Albicans in root -

filled teeth with apical periodontits of type - 2 diabetic patients using spectroscopy.

C. Comparative microbial analysis of the above two groups.

D. Signature library construction for Enterococcus faecalis , Streptococcus Species, and

Candida Albicans .

E. To investigate the association of the above species with specific endodontic signs and

symptoms.

7. MATERIAL AND METHODS:

7.1 Source of the Data:

Root canal samples from 40 subjects, 20 subjects with non diabetic and 20 diabetic

subjects 25-75 years of age with secondary root canal infection referred for non surgical

endodontic retreatment to KVG Dental college and Hospital, Sullia, will be included for the

study.

7.2 Method of Collection of Data

Informed Consent and Ethical Clearence

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.

Data collection and Sampling size

Clinical root canal samples of 40 subjects, 20 non diabetic and 20 type-2 diabetic subjects 25-

75 years of age with a diagnosis of apical periodontitis requiring endodontic retreatment will be

divided into 2 groups . All 40 subjects will be screened for Diabetes mellitus.

Group 1 (20 No’s) - Healthy Non diabetic subjects with secondary root canal infection

requiring retreatment.

Group 2 (20 No’s) – Type-2 diabetic subjects with secondary root canal infection requiring

retreatment.

WHO criteria for diabetes mellitus - RBS > 200 mg/dl or FBS > 126mg/dl, or 2hr PPBS > 200

mg/dl or glycosylated HB > 7% will be assessed.

For each sample, detection of Enterococcus faecalis , Streptococcus Species and Candida

Albicans will be done using spectroscopy in Indian institute of science, Bangalore.

Inclusion Criteria

1. Patients aged 25-75 years.

2. Both males and females will be included.

3. Patient requiring retreatment of endodontically treated teeth with a diagnosis of apical

periodontitis and those willing to participate in the study.

4. Patients who had undergone endodontic therapy more than 3 years ago.

5. Radiographic evidence of periradicular disease.

6. The termini of the root canal fillings should be atleast 2mm short of the radiographic apex.

7. The subjects in group 2 will have a random blood sugar level > 200mg/dl, Fastiing blood

sugar level >126mg/dl, 2hr Post prandial blood sugar level > 200mg/dl and glycosylated

HB > 7%.

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. Use of any antibiotics in past 3 months

4. Teeth that cannot be isolated with rubber dam

5. Calcified canals

6. Tortuous canals

7. Root fracture

8. Teeth with developmental defect.

Sample Selection

Case history which comprises of detailed History, clinical feature and radiographic feature

will be recorded for all 40 subjects.

The presence or absence of following features will be evaluated to associate it with the

species of microorganism detected for every sample.

Pain.

Intensity- Mild, moderate, Severe

Frequency-Intermittent to continuous pain

Tenderness to percussion

Coronal restoration

Acute, chronic or subacute lesion.

Mobility

Size of periapical lesion

Swelling

Sinus or Fistula

Foul Odour

Wet or Dry canal

Periodontal status of the tooth

Radiographic quality of the root canal filling

Apical extent of root canal filling

Armamentarium and Materials

1 .Diagnostic Instruments: Mouth Mirror, Explorer, Tweezer.

2. X-rays

3. Lignocaine with Adrenaline

4. Disposable Syringes

5. Rubber dam

6. Spoon Excavator

7. Airotor handpiece

8. Micromotor handpiece

9. Anthogyr handpiece

10. Access cavity burs

11. Pumice

12. 30% hydrogen peroxide

13. 2.5% sodium hypochlorite solution

14. 5% sodium thiosulphate

15. #15 K-type file

16. Paper points

17. 0.9% Sterile Saline solution

18. Eppendorf tubes

19. Test tubes

20. RTF solution

22. Petri Dishes

22. Blood agar

23. Mitis-Salivarius agar

24. Mac-Conkey’s agar

25. Sabournaud’s dextrose agar

26. Sterile wire loop

27. Candle jar

28. Incubator

29. Pure freezed culture of Enterococcus faecalis , Streptococcus Species and Candida Albicans

obtained from MTCC, Chandigarh.

30. Aluminium coated microscopic slide.

31. Spectroscopy detection system

Root Canal Retreatment and Sampling Procedure

All retreatment 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 will be achieved with a sterile high speed

carbide bur until the root filling will be exposed. 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 5% sodium thiosulfate solution to inactivate the NaOCL .

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 occurrs, the patient sample will be disqualified from the study.

Coronal gutta-percha will be removed by using Gates-Glidden drills; apical material will

be retrieved with Hedstrom files.The working length determination will be done by

radiographic and electronic method.

Two sequential sterile paper points will be placed till working length for 1 min to soak

up the fluid in the canal and transferred to the sterile tube containing 0.75 mL of RTF solution.

Bacterial Cell Preparation and Growth conditions

The culturing procedure will be carried out in Department of microbiology, K.V.G Medical

College and Hospital, Sullia. Tubes containing the transport medium will be shaken in a vortex

mixer for 60 seconds. Samples will be plated onto Blood agar and different selective media

using sterile wire loop:

Mitis-Salivarius agar: Streptococcus mitis

Mac Conkey’s agar: Enterococcus faecalis

Sabournaud’s dextrose agar: Candida albicans

The inoculated culture media will be incubated as follows:

Streptococcus mitis: candle jar.

Enterococcus faecalis: Aerobic technique

Candida albicans : Aerobic technique

Incubation will be done at 370C for 48 hours.

The growth suspected to be Streptococcus mitis/Enterococcus faecalis/Candida albicans based

on phenotypic characteristics on Blood agar and their respective Selective media will be

subcultured on Blood agar.

Signature library construction for Enterococcus faecalis, Streptococcus Species, and

Candida Albicans.

A comprehensive pathogen signature library will be constructed using spectroscopy

technology for Enterococcus feacalis , Streptococcus Species, and Candida Albicans ,the most

common endodontic microorganisms in secondary root canal infection . For this pure freezed

culture of these bacteria will be obtained from MTCC, Chandigarh. Samples of pure culture will

be transferred to aluminium coated microscopic slides and scanned using spectroscopy. The

signatures must be reproducible and must exhibit minimal variability induced by sensor

uncertainity, biological growth conditions, and sample preparation and dissermination

condition. The signatures obtained are used as a reference for pathogen detection .

Evaluation of microorganisms by Spectroscopy.

1.0-cm diameter portions of agar with nondisturbed bacterial growth from each species will be

transferred to a Aluminium coated microscopic slide and scanned using spectroscopy. The

graph obtained is compared with the endodontic pathogen library and Data analysis will be

done.

Follow up: Nil

Follow up period: Nil

Analysis of data

Analysis will be done using Chi-square test.

Flow chart of Investigation design:

40 subjects (20 non diabetic and 20 type-2 diabetic subjects) with secondary root canal infection

referred for non surgical endodontic retreatment to KVG Dental college and Hospital, Sullia,

will be included for the study.

Informed consent from all patients will be taken

Charting of records - medical and dental history

History Clinical features Radiographic features

Root Canal Retreatment and Sampling Procedure

Bacterial Cell Preperation and Growth conditions

Signature library construction for Enterococcus faecalis, Streptococcus Species,and Candida

Albicans.

Evaluation of microorganisms by Spectroscopy

The graph obtained is compared with the endodontic pathogen library and

Data analysis will be done using Chi-square test.

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 40 subjects. The study does not inflict any harm to the patients .This study will be done

under the supervision of my guide

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

Yes. (attached document no:2)

8 . REFERENCES

1. Gomes P F A,Ericka T. Pinheiro Rogério C. Jacinto,Alexandre A. Zaia,Caio C.R. Ferraz, Francisco J. Souza.Microbial Analysis of Canals of Root-filled Teeth with Periapical Lesions Using Polymerase Chain Reaction.JOE 2008;34(5):537-30.

2. Khedmat S .Evaluation of endodontic treatment failure of teeth with periapical radiolucent areas and factors affecting it. Journal of Dentistry, Tehran University of Medical Sciences 2004;1(2):34-38.

3. Undqvist G.S, Figdor D.Life as an endodontic pathogen Ecological differences between the untreated and root-filled root canals.Endodontic Topics 2003; 6:3–28.

4..Bender B, Bender A. B. Diabetes Mellitus and the Dental Pulp.JOE. 2003; 29(6): 383-89.

5. Mohan.V, Sandeep.S, Deepa.R “Epidemiology of type 2 diabetes:Indian scenario” Indian J Med Res125, March 2007, 217 – 30.

6. Ramachandran A, DAS A.K, Joshi S.R, Yajnik C.S, Shah S, Kumar P. Current Status of Diabetes in India and Need for Novel Therapeutic Agents. Supplement To Japi. 2010 june ; 58:7-9.

7. Kalasinsky K.S, Hadfield.T, Shea.A, Kalasinsky V.F, Nelson M.P, Neiss J, Drauch.A.J, Vanni.G.S, Treado P.J. Raman Chemical Imaging Spectroscopy Reagentless Detection and Identification of Pathogens: Signature Development and Evaluation. Anal Chem 2007; 79: 2658-2673.

8. Howell S.C, Haffajee A.D, Pagonis.T.C, Guze.K.A. Laser Raman Spectroscopy as a Potential Chair-side Microbiological Diagnostic Device.JOE.2011; 37: 968-72.

9. Schirrmeister J F, Priv-Doz , Liebenow A L, Pelz K, Wittmer A, Serr A, Hellwig E, Al-Ahmad A. New bacterial compositions in root filled teeth with periradicular lesions, JOE 2009;35:169-174.

10. Lin L.M, Skribner J E, Gaengler P. Factors Associated with Endodontic Treatment Failures.Journal of Endodontics.1992;18(12):625-627.

11. Molandera A, Reita C,Dahlénb G,Kvista T. Microbiological status of root-filled teeth with apical periodontitis.Int Endod J. 1998;31:1-7.

12. Fouad AF. "The effect of diabetes mellitus on endodontic treatment outcome" JADA 2003 ; 134 : 43-51.

13. Segura-Egea J.J, Jimenez-Pinzon A, Rios-Santos J.V, Velasco-Ortega E, Cisneris-Cabellos R, Povato-Ferrara M. "High prevalence of apical periodontitis among type 2 diabetic patients" Int Endod J . 2005 ; 38 : 564-69.

K. V. G. DENTAL COLLEGE & HOSPITAL (Document No-1)

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

SPONSORED BY ACADEMY OF LIBERAL EDUCATION (REGD.) SULLIA

Department of Conservative Dentistry and EndodonticsDepartment of Conservative Dentistry and Endodontics

INFORMED CONSENT FORM

Name of the participant: ____________________________________________

Name of the Investigator: ______________________________

Name of the Institution: ____________________________________________

Documentation of the informed consent

I, … … … … … … … … … ., have read the information in this form (or it has been read to me). I was free to ask any questions and they have been answered. I am over 18 years of age and, exercising my free power of choice, hereby give my consent to be included as a participant in “… … … … … .… … … … … … … … … … … … … … . … … … … … … … … … … … … … ” (title of the study)

I,here by authorize Dr. ___________________ and any other agents or employees of

______________________________________ and such assistants as may be selected by any of them for

the study described.

Consent : Written Oral

(1) I have read and understood this consent form and the information provided to me.

(2) I have had the consent document explained to me in understandable language.

(3) I have been explained about the purpose and procedure of the study and that the study involves research.

(4) My rights and responsibilities have been explained to me by the investigator.

(5) I have been advised about the risks associated with my participation in the study.

(6) I have informed the investigator of all the treatments I am taking or have taken in the past.

(7) I agree to cooperate with the investigator and I will inform him/her immediately if I suffer unusual symptoms.

(8) I have not participated in any research study within the past 6 months.

(9) I am aware of the fact that I can opt out of the study at any time without having to give any reason and this will not affect my future treatment in the hospital.

(10) I am also aware that the investigators may terminate my participation in the study at any time, for any reason, without my consent.

(11)I have been informed of possible alternative methods of treatment including no treatment at all.

(12)I have been given the opportunity to question the doctor concerning the nature of treatment, the

inherent risks of the treatment, and the alternatives to the treatment.

(13)I will visit the Dentist as and when required for the study, at the given appointment.

(14)I permit the operator to utilize the information given by me and results obtained from the study for presentation and publications.

(15)I will follow the instructions given by the doctor during the study

(16)It has been explained to me and I understand that perfect results is not guaranteed or warranted and cannot be guaranteed or warranted.

(17) I have had my questions answered to my satisfaction.

(18) I have decided to be in the research study voluntarily.

Patient's Name:

Address:

Phone No:

Signature:

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: ___________________

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.

PROFESSOR AND H.O.D, 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. MOKSHA NAYAK M.D.S.

PROFESSOR AND H.O.D, 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