Comparative Evaluation of Bacterial Culture and Polymerase Chain Reaction for Detection

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE- KARNATAKA

ANNEXURE- II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

1. NAME OF THE CANDIDATE
AND ADDRESS / DR.NITHYA PUSHPANATHAN
POST GRADUATE STUDENT
DEPT OF CONSERVATIVE & ENDODONTICS
K.V.G DENTAL COLLEGE & HOSPITAL KURUNJIBAGH
SULLIA- 574327
2. NAME OF THE INSTITUTION / K.V.G DENTAL COLLEGE & HOSPITAL
3. COURSE OF STUDY & SUBJECT / MASTER OF DENTAL SURGERY IN CONSERVATIVE DENTISTRY & ENDODONTICS
4. DATE OF ADMISSION TO COURSE / 07-06-10
5. TITLE OF THE TOPIC / COMPARITIVE EVALUATION OF BACTERIAL CULTURE AND POLYMERASE CHAIN REACTION FOR THE DETECTION OF ENTEROCOCCUS FAECALIS IN RETREATMENT CASES WITH APICAL PERIODONTITIS

COMPARATIVE EVALUATION OF BACTERIAL CULTURE AND POLYMERASE CHAIN REACTION FOR DETECTION OF ENTEROCOCCUS FAECALIS IN RETREATMENT CASES WITH APICAL PERIODONTITIS

6. BRIEF RESUME OF THE INTENDED WORK

6.1 Need for the Study:

A great deal of scientific evidence indicates that microorganisms involved in Intraradicular or Extraradicular infections are the major causative agents of endodontic therapy failure1.Bacteria or their products are considered to be the primary aetiological agents of pulpal necrosis and periapical lesions2 and it has an unequivocal role as a causative agent of apical periodontitis3. Persisting bacteria in root canals maybe those originally present in the necrotic pulps that survive the biomechanical procedures3.

The frequency of E. faecalis found in persistent periradicular lesions has been shown to be much higher. In fact, failed root canal treatment cases are nine times more likely to contain E. faecalis than primary endodontic infections.Studies investigating its occurrence in root-filled teeth with apical periodontitis have demonstrated a prevalence ranging from 24 to 77%4 . They are known to have a low susceptibility to Ca(OH)25 and also show resistance to the antimicrobial effects of calcium hydroxide, probably due to an effective proton pump mechanism which maintains optimal cytoplasmic pH levels6. In in vitro studies, E. Faecalis has been shown to invade dentinal tubules whereas not all bacteria have this ability. E. faecalis, moreover, can enter the viable but non-cultivable (VBNC) state, a survival mechanism adopted by a group of bacteria when exposed to environmental stress, and resuscitate upon returning to favourable conditions6.

Traditionally, endodontic bacteria have been studied by means of cultivation-based techniques. The main advantage of cultivation approaches are related to their broad- range nature, which makes it possible to identify a great variety of microbial species in a sample, including those that are not being sought after. However, cultivation and other traditional identification methods have been demonstrated to have several limitations when it comes to microbiological diagnosis. Some of the reasons for bacterial unculturability are the lack of essential nutrients or growth factors in the artificial culture medium, toxicity of the culture medium itself, which can inhibit bacterial growth and production of substances inhibitory to the target microorganism by other species present in a mized consortium8.

Novel culture- independent methods for microbial identification that DNA amplification of 16S rDNA followed by sequencing has recently been used to investigate the microbial diversity.They are more specific, accurate, sensitive and rapid than culture technique, and can detect uncultivable and fastidious microorganisms. It is a technique, which uses a DNA polymerase enzyme to make a huge number of copies of virtually any given piece of DNA or gene. Most PCR assays doesnot quantitatively detect the microorganism and false positive results have the potential to occur because of PCR amplification of contaminant DNA. False negative may occur due to presence of enzyme inhibitors or nucleases in clinical samples8.

The prevalence of some oral pathogens varies among subjects of various ethnic backgrounds.Differences in environmental and genetic determinants of microbial colonization of the oral cavity may be responsible for varying occurrence of some endodontic pathogens in different populations10.

The lack of data from the Indian population regarding this analysis adds to the significance of this study and hence the overall aim is to compare and analyse the presence of E. faecalis from retreatment cases, using culture and polymerase chain reaction methods.

6.2 Review of Literature:

A study was done to investigate the occurrence of Enterococcus faecalis in root canals of previously root filled teeth with apical periodontitis requiring retreatment in Lithuanian patients. Twenty five asymptomatic teeth were included in the study. Avoiding contamination, microbiological samples were taken from the canals before and after preparation and irrigation with sodium hypochlorite and EDTA. E. faecalis was isolated from 14 of those 20 culture positive teeth. Second samples taken after preparation revealed growth in 7 of the 20 teeth. The study concluded that, rather than previous chemical treatment, it is the ecological conditions present in the incompletely filled root canal that are important for the presence of E. faecalis in these teeth7.

A study was done to investigate the composition of the microbial flora present in teeth after the failure of root canal therapy in a North American population and these results were compared with the previous Scandinavian studies. 54 root-filled teeth with persistent periapical radiolucencies were selected for retreatment. Enterococcus faecalis was the most commonly recovered bacterial species and was identified in 30% of the teeth with positive cultures. This study concluded that bacteria was cultivable at rates similar to those seen in the Scandanavian studies6.

A study was done to identify the microbial flora within root canals of teeth with failed root-canal treatment and to determine the association of the various species with clinical features. Sixty root-filled teeth with persisting periapical lesions were selected. Significant associations were observed between pain and polymicrobial infections or anaerobes, tenderness to percussion and Prevotella intermedia, sinus and Streptococcus spp. or Actinomyces spp. and coronally unsealed teeth and Streptococcus spp. or Candida spp. Of the microbial species isolated, 57.4% were facultative anaerobic species and Enterococcus faecalis was the most frequently recovered bacterial species3. It concluded that the microbial flora in canals after failure of root canal treatment were limited to a small number of predominantly gram positive microbial species2.

A study was done to investigate occurrence of micro-organisms in cases of failed endodontic treatment in the Brazilian population, by means of the polymerase chain reaction (PCR). Samples were taken from 22 root-filled teeth with persistent periradicular lesions selected for retreatment and the DNA was extracted from the samples and analyzed. Microorganisms occurred in all cases of root filled teeth associated with periradicular lesions, which supports the assertion that treatment failures are rather of infectious etiology, caused by persistent or secondary intraradicular infections. E. faecalis was the most prevalent species, detected in 77% of the cases, followed by P. alactolyticus, P. propionicum, D.pneumosintes and F alocis1

A study was done to assess the occurrence of nine putative endodontic pathogens in root filled teeth associated with periradicular lesions in a South Korean population using culture independent molecular approach. Fourteen root filled teeth with persistent periradicular diseases were selected for retreatment. Bacteria was present in all cases, as revealed by amplification using ubiquitous 16S rDNA primers.The most frequently detected taxon was Enterococcus faecalis (64%) followed by Streptococcus spp: (21%) and Tannerellaforsynthensis (14%). This study strengthen the assertion that intraradicular infections are the main cause of endodontic failures and that E.faecalis is a common member of the microbiota associated, regardless of geographical location10.

A study was done to identify Enterococcus spp. in nonhealing endodontic cases using PCR and molecular sequencing, and also to determine if the prevalence of enterococcus is increased in diabetic patients. 37 specimens were incubated in prereduced thioglycollate broth at 370C. 8 specimens were positive for Enterococcus spp. of which 19% were from non diabetic and 33% from diabetic patients. Enterococcus faecalis was the sole enterococcal species detected with an overall prevalence of 22% and was found more in diabetic patients. These organisms were found to be resistant to antibiotics except vancomycin11.

A study was done to compare real-time quantitative PCR (qPCR) assay to cultivation for the detection of Enterococcus faecalis and quantitation during endodontic therapy. A reverse transcription PCR (RT-PCR) assay was also developed to detect the bacterium clinically in the VBNC state. 87 samples were collected upon access, post instrumentation/irrigation and postcalcium hydroxide treatment from 15 primary and 14 refractory infections involving 29 single-rooted teeth, and analysed. Overall, qPCR and RT-PCR were found to be more sensitive than cultivation for identifying Enterococcus faecalis in endodontic infections. It also demonstrated that the bacterium, likely in the VBNC state; can persist after therapy5.

A study was done to investigate the presence of Enterococcus faecalis in endodontic infections in deciduous and permanent teeth by culture and PCR methods. Among the 145 molar teeth selected, 57% presented necrotic asymptomatic pulp tissues. Enterococcus faecalis was cultured from 18% of necrotic deciduous teeth and 26% of necrotic permanent teeth. PCR detection identified the target species 22% and 32% of deciduous and permanent teeth respectively4.The results of this study confirmed that both culture and PCR methods are sensitive to detect E. faecalis in root canals4.

6.3 Aim and objective of the Study:

Aim:

To compare bacterial culture andpolymerase chain reaction for the detection of enterococcus faecalis in retreatment cases with apical periodontitis in rural Indian population.

Objectives:

To evaluate the effectiveness of bacterial culture for the detection of enterococcus faecalis in retreatment cases with apical periodontitis.

To evaluate the effectiveness of polymerase chain reaction of enterococcus faecalis in retreatment cases with apical periodontitis.

Tocompare bacterial culture and polymerase chain reaction for the detection of enterococcus faecalis in retreatment cases with apical periodontitis.

To co-relate the signs and symptoms with presence of Enterococcus faecalis.

7. MATERIALS AND METHODS

7.1 Source of the Data

Clinical samples from 40 subjects (men and women, 18-75years of age) referred for nonsurgical endodontic retreatment, to K.V.G Dental College and Hospital, Sullia, will be included for the study.

The study will be initiated subsequent to approval of ethical committee.Consentsof patients willing to participate in the study will be obtained in a given format.A detailed medical and dental history will be obtained from each patient.

Diagnosis of secondary endodontic infection:

1) Case history:

Previous history of endodontic treatment

The symptoms include

Pain
Intensity:Moderate to severe pain
Frequency:Intermittent or continous pain

2) Clinical examination:

endodontically treated tooth
Tooth tender on percussion
there may or may not be mobility of involved tooth
Vitality tests gives a negative response

3) Radiographic examination:

root filled tooth
Periapical radiolucency less than 4 mm
Widened PDL space
the termini of the root canal fillings ranged from 0 to 5mm short of the radiographic apex.

Pre-operative assessment of periapical status will be done with CPDR, QLDR and QTDR criteria as follows:

A) CPDR criteria (Clinical periapical diagnosis of root

Normal

No clinical symptoms or

Periradicular sclerosis

Acute

Clinical signs and symptoms of inflammation.

Periradicular sclerosis or rarefaction ≤ 1mm visible on radiograph

Chronic

No clinical signs and symptoms of inflammation

Periradicular rarefaction > 1mm

Exacerbating

Clinical signs and symptoms of periradicular

Inflammation, infection, or both.

Periradicular rarefaction > 1mm

B) QLDRcriteria (Qualitative radiographic diagnosis of tooth)

Normal periapex / No radiographically discernible pathosis, except for widened periodontal ligament
Diseased periapex / Presence of any discernible periapical radiolucency

C)QTDRCriteria (Quantitative radiographic diagnosis of tooth)

Normal Periapex / No radiographically discernible pathosis, except for widened periodontal ligament with intact lamina dura
Acute apical periodontitis / Periapical radiolucency is present and measures
≤ 1 mm.
Chronic resorbing and exacerbating apical periodontitis / Periapical radiolucency largest is >1mm

7.2 Method of collection of data (including sampling procedure, if any)

The samples will be divided into 2 groups.

Group1(40 Nos.): Detection of Enterococcus faecalis in patients of retreatment cases with apical periodontitis, using culture method

Group2(40 Nos.): Detection of Enterococcus faecalis in patients of retreatment cases with apical periodontitis, using PCR method.

I)Inclusion Criteria:

Patients aged 16-75 years.
Both males and females will be included.
Patient requiring retreatment of endodontically treated teeth with a diagnosis of apical periodontitis in any of the teeth.
Patients who had undergone endodontic therapy more than 2 years earlier.
Termini of the root canal fillings areatleast 2mm short of the radiographic apex.

II)Exclusion criteria:

Systemic diseases
Pregnancy and lactation
Use of any antibiotics in past 3 months
Participation in other clinical study during previous 3 months
Teeth that can not be isolated with rubber dam
Teeth exhibiting frank exposure of the root filling material to the oral cavity.
Calcified canals
Tortuous canals
Root fracture
Teeth with developmental defects

III)Armamentarium and Materials.

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

Lignocaine with Adrenaline
Disposable Syringes
Rubber dam
Spoon Excavator
Airotor Hand Piece
Micromotor Hand Piece
Anthogyr gear handpiece
Access cavity burs
Pumice
30% hydrogen peroxide
2.5% sodium hypochlorite solution
5% sodium thiosulphate
#15 K-type file
#15 file
Paper points
Saline solution
Eppendorf tubes
Test tubes
Petri dishes
Blood agar
Mac Conkey’s agar
RTF solution
Crystal violet
Carbolfuschin
Gram’s iodine
Acetone/ Alcohol

25. 38 μl sterile distilled water

26. 5 μl 10X PCR buffer (500 mM KCl, 100 mM Tris-HCl (pH 9.0),

1.0% Triton X 100)

27. 3 μl 25 mM MgCl2

28. 1 μl 10 mM dNTPs (10 mM each dATP, dTTP, dGTP. dCTP)

29. 1 μl 20 μM forward primer

30. 1 μl 20 μl reverse primer

31. 0.2-1 μl Taq polymerase

32. 50 μl total volume

33. Eppendorf Master cycler

34. Eppendorf Centrifuge 5415-D

35. Eppendorf Pipette

36. Eppendorf Pipette tips

37. 100BP DNA Ladder from NEB Company (Catalog No: N3271)

IV)INVESTIGATION DESIGN:

40 subjects with diagnosis of apical periodontitis in root filled teeth.

Informed consent from all patients will be taken

Charting of records - medical and dental history

Diagnosis of apical periodontitis will be done by

Each tooth will be cleansed with pumice and isolated using rubber dam

Tooth and surrounding field will be cleansed with 3% hydrogen peroxide and decontaminated with 2.5% sodium hypochlorite.

After plaque removal, isolation, and disinfection of the operative field, the coronal restorations will be removed.Endodontic access will be completed with a sterile high-speed carbide bur until the root filling is exposed.

If a post is present, removal will be attempted through ultrasonic vibration; if this is unsuccessful, post removal will be carried out with a sterile high-speed carbide bur.

After completion of the endodontic access, the tooth, clamp, and adjacent rubber dam will be disinfected with 2.5% NaOCl. The cavity will be swabbed with 5% sodium thiosulphate solution to inactivate the NaOCl.

Coronal gutta-percha will be removed by means of sterile Gates-Glidden burs, and the apical material retrieved using K-type or Hedstrom files, or both.

Removal of root fillings will be performed without the use of chemical solvents. Whenever possible, filling material removed from the canals will be transferred to tubes containing RTF solution toensure that all filling materials is removed.

A small amount of sterile saline solution will be introduced into the root canal via syringe, and the canal walls will be filed so that material is obtained

Working length of the canal will be determined radiographically using #20 K-file.The root canal will be prepared with a #20 K-file 0.5-1.0mm short of the radiographic working length.

2 paper points will be placed in the root canal. Each paper point will be retained in position for 60sand the samples will be transferred to sterile eppendorf tubes containing RTF transport medium(2ml).

Samples will be subjected to

All samples will be processed within 2 hrs. After thoroughly shaking

the endodontic sample in a mixer for 60 s, 1 ml of each sample will be used for culture and the other 1ml samples will be frozen immediately at -20oC andstored until assayed by PCR.

Statistical analysis using Chi-square test

Will be done

V)METHODOLOGY

Culture experiments will be performed in the Microbiology Department of K.V.G. Medical College, Sullia.
PCR Techniques will be carried out in National Institute Of Biological Sciences, Tata Institute Of Fundamental Research, Bangalore.

Culture Identification

The root canal samples obtained will be immediately placed in reduced transport media and then submitted to microbiology laboratory.

Reduced transport media contains 1mmol/L EDTA,3.7mmol/L Na2CO3,2.5mmol/L K2HPO4,15mmol/LNaCl,6mmol/L(NH4)2SO4,3mmol/L KH2PO4,1mmol/L MgSO4.

Tubes containing the transport medium will be shaken in a vortex mixer for 60 seconds.0.01ml of sample will be transferred and plated on Mac Conkey’s agar and Blood agar plates using calibrated inoculation wire. The plates will be incubated aerobically for 24hrs and the E. faecalis counts will be determined semiquantitatively. The purity of the cultures will be confirmed by Gram staining, catalase production, colony morphology on the agar and using standard biochemical analysis.

Identification of bacteria

After incubation,each plate will bebiochemically analysed for growth and identification of bacteria using the colony morphology,growth in specific media,enzymatic activities and fermentation of sugars

Enterococcus faecalis

Magenta red coloured colonies in Mac Conkey agar.

Gram stain shows gram positive pairs of oval cocci,the cells in a pair arranged at an angle to each other.

Non-haemolytic colonies in blood agar..

Growth at 6.5% NaCl.

Growth on bile-esculin agar.

Growth at 45oC.

Black colonies on Bile esculin agar.

Fermentation of mannitol,sucrose,sorbitol.

DNA Extraction

Samples in RTF will be thawed to 37oC for 10min and vortexed for 30s. The pellets will be then resuspended in redistilled water, boiled for 10min and chilled on ice. After centrifugation, the supernatant will be collected and used as a template for PCR amplification. DNA is extracted and would be used as positive control for the primers. The strain would be supplied by Sigma Aldrich Laboratory, Bangalore.

PCR primers, with expected amplicon size and thermocycling parameters used in the present study are 5’- GTT TAT GCC GCA TGG CAT AAG AG-3’(forward primer) and 5’-CCG TCA GGG GAC GTT CAG-3’(reverse primer). Negative controls consisting of ultrapure waterinstead of sample will be included with each batch of samples analyzed.