RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA
BANGALORE.
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / Name of the candidate and address(in block letters) /
DR. SNEHALATA PATIL
POST-GRADUATE STUDENTDepartment OF CONSERVATIVE DENTISTRY AND ENDODONTICS
AME’s DENTAL COLLEGE & HOSPITAL
BEJENGERE ROAD, RAICHUR – 584 103
2. / Name of the Institution / AME’s DENTAL COLLEGE, HOSPITAL AND RESEARCH CENTRE
BEJENGERE ROAD, RAICHUR – 584 103
3. / Course of study and subject / MASTER OF DENTAL SURGERY (MDS)
CONSERVATIVE DENTISTRY AND ENDODONTICS
4. / Date of admission to course / 29-05-2010
5. /
TITLE OF THE TOPIC
COMPARATIVE EVALUATION OF ANTIFUNGAL EFFICACY OF 3% SODIUM HYPOCHLORITE, 2%CHLORHEXIDINE GLUCONATE, AND 17% EDTA WITH AND WITHOUT ANTIFUNGAL AGENTS – AN IN VITRO STUDY6.
7. / Brief resume of the intended work
6.1 Need for the study
Microbes are associated virtually with all diseases of the pulp and periradicular tissues. The
microbial ecosystem of infected root canals may consist of bacteria, including spirochetes and fungi.1 Fungi are common opportunistic pathogens that constitute a part of normal microbial flora of the oral cavity. They are also detected in 25% of root canal.
Candida is a versatile microorganism capable of adapting itself to a wide range of pH levels. It exhibits a variety of virulence factors such as adherence, thigmotropism and phenotypic switching and secretes a degenerative enzyme “aspartyl protease” that degrades the dentinal collagen. It is considered as a “dentinophillic” microorganism and has the ability to grow on the dentinal surfaces in the absence of oral tissue fluids and penetrates in to the dentinal tubules by its various growth patterns. The higher rate of protease activity of candida albicans in comparison with other candida species suggests its higher virulence.2
Microbial control within the pulpal tissue and root canal space is a prerequisite for the prevention and treatment of pulpal and periapical breakdown. So the ideal irrigant to eliminate microorganisms should be strongly antimicrobial but should not be toxic to the periapical tissues.3
In a poorly functioning immune system the risk of fungal infection might increase in root canal spaces.4Therefore,the disinfection of the root canal should incorporate an antifungal agent during cleaning and shaping procedures as an adjunct along with other endodontic irrigants like sodiumhypochlorite, chlorhexidine gluconate, and ethylenediamine tetra acetic acid [EDTA].
6.2 Review of literature
A study was conducted to evaluate the occurrence of candida albicans in infections of endodontic origin using the polymerase chain reaction (PCR). Twenty four samples were taken from infected root canals and ninghteen aspirates from periradicular infections of endodontic origins. Result showed the presence of candida albicans in five of twenty four (21%) samples taken from root canals, but none was detected in the periradicular aspirates.1
A comparative study was conducted on sixty five extracted human mandibular teeth to evaluate the antifungal efficacy of 5.25% sodium hypochlorite, 2% chlorhexidne gluconate, and 17% EDTA with and without antifungal agent. The samples of group 1 were irrigated without any antifungal agents where as samples of group 2 were irrigated with an antifungal agent along with other irrigants. Statistical analysis showed that, the inclusion of 1% clotrimazole along with the experimental irrigants further enhanced the antifungal efficacy.2
A study was undertaken to evaluate the antimicrobial effect of six root canal irrigants on selected microorganisms. Irrigants used were 5.25% NaOCl, 0.5% NaOCl, 2% chlorhexidine, 21% alcohol, cresophene and sterile saline solution. Microorganisms were sub cultured on brain heart infusion agar (BHI) in 5% sheep blood agar and BHI agar. Several colonies from each plate were taken and incubated. Then plates were swabbed with the bacteria suspension. Paper discs were soaked with test solutions and placed on the
plates and re-incubated. Zones of Inhibition were measured. Tests were repeated five times for all strains. Statistical analysis was carried out using an ANOVA with Duncan’s test of multiple comparisons and concluded that, 5.25% NaOCl was superior in its antimicrobial abilities and reduced concentration of NaOCl decreases its effect when compared with other irrigants used.3
A study was conducted on forty-eight single rooted teeth to compare the antifungal efficacy of 6% sodium hypochlorite, 2% chlorhexidne gluconate, 17% EDTA and BioPure MTAD (Mixture of Tetracycline Acid and Detergent) as a final rinse on candida albicans. After root canal preparation, teeth were inoculated with Candida albicans. Aliquots from the experimental teeth were plated on Sabouraud 4% dextrose agar plates and colony-forming units were counted as a measure of antifungal activity. They concluded that 6% sodium hypochlorite and 2% chlorhexidne gluconate were equally effective and statistically superior to Bio-Pure MTAD and 17% EDTA in antifungal activity.4
A study compared the antimicrobial effect of NaOCl, MTAD(Mixture of Tetracycline Acid and Detergent) and their mixture against resistant microbes in root canal therapy with Zone of inhibition (ZI) with 150 specimens and Minimum Inhibitory Concentration (MIC) with 155 tubes in vitro. The microbes tested were Enterococcus faecalis, Pseudomona aeruginosa, Escherichia (E) coli, Staphylococcus aureus and Candida albicans. Each microbes are incubated in different media and after 24 hours a suspension was prepared
from each microbial species with a concentration of 1 McFarland and used in the investigation. In ZI technique blood agar plates were inoculated with organisms, paper discs were soaked with irrigants with maximum zones of bacterial inhibition were recorded. In MIC technique the irrigants were serially diluted in Trypticase Soy Broth (TSB) tubes and 0.1 mL of the tested microbe solutions were added. Results were obtained on the basis of turbidity and growth on agar plates. Statistical analysis was carried out using ANOVA and Tukey tests. In ZI technique, the results demonstrated that MTAD has greater antimicrobial efficacy compared to NaOCl, and their mixture (M+N) against E. faecalis, S. aureus, and Enteric bacteria. NaOCl was more effective in eradicating C. albicans than the others. In MIC technique, MTAD was more effective against S. aureus, and E. bacteria. MTAD and NaOCl were equally effective against E. faecalis however; NaOCl was more effective against C.albicans. So they concluded that bacterial species were more susceptible to MTAD than NaOCl. C.albicans, however more susceptible to NaOCl due to broad spectrum antimicrobial activity. So the mixture of MTAD and NaOCl did not prove to be more effective than their individual use.5
A study was conducted on 266 extracted human incisor teeth, to evaluate the antifungal properties of 0.12% chlorhexidine, 1% and 5% NaOCl. Experimental root canals dispensed with C. albicans inoculum were irrigated with 3ml of either disinfected solutions for 1min, 5mins, 30mins, and 1hour followed by incubation in test tubes having Sabouraud’s Dextrose Broth at 370 for 24 hours. It was concluded that antifungal activity was observed in 1hour for all solutions in the presence of smear layer but in the absence of smear layer, 5% NaOCl alone started to show antifungal activity after 30 mins.6
6.3 Objectives of the study
To evaluate
1) The antifungal efficacy of 3% sodium hypochlorite, 2% chlorhexidine gluconate, and 17% ethylenediamine tetra acetic acid with and without antifungal agent.
2) To compare the antifungal efficacy of Clotrimazole and Nystatin against candida albicans.
Material and methods
7.1 Source of data
Materials used:
1. Endodontic irrigants:
a. 3% Sodium hypochlorite (NaOCl)
b. 2% Chlorhexidine gluconate
c. 17% EDTA
2. Antifungal agents
a) Clotrimazole- 10mg/mL
b) Nystatin- 1.5 mg/mL
3. Single rooted anterior teeth
4. Ultrasonic scaler
5. 0.2% Sodium azide to store the teeth
6. Barbed broaches
7. 25mm, size 15K file
8. Gates Glidden drills 1 to 4 no.
9. Distilled water
10. Sterile saline
11. Type II GIC
12. Autoclave to sterilize the roots
13. Glass test tube to store the experimental teeth
14. Suspension of Candida albicans
15. Humidor
16. 4% Sabouaraud dextrose agar plate to verify the growth of Candida albicans
17. Sterile absorbent paper points to dry the canals
18. Inoculation loop to remove aliquots
19. Incubator
20. Light microscope to assess the growth of Candida albicans
7.2 Method of data collection:
One hundred eighteen extracted human, single rooted, anterior teeth will be immersed in 3% sodium hypochlorite for 15mins to remove debris and organic tissues and will subsequently be cleaned using an ultrasonic scaler to render them free from calculus and tissue tags. They will be stored in 0.2% sodium azide until used.
All the teeth will be decoronated at the cementoenamel junction, and pulp remnants will be extirpated using barbed broaches. A 25mm, size 15K-file will then be inserted into the root canal until it is seen out at the apical foramen. 1mm will be subtracted from that measured length and will be considered as working length. Gates Glidden drills (1 to 4) will be used for the coronal flaring, and apical preparation will be done until ISO size 50. 3mL of 5.25% NaOCl will be used between filing. After instrumentation, the removal of smear layer will be accomplished with a final rinse of 1mL of 17% EDTA for 1min followed by 3mL of 5.25% NaOCl. Finally the canal will be flushed with 5mL of distilled water to remove any residual debris and irrigants. The root will be coated with two coats of nail varnish and apical foramen will be sealed with type II GIC.
The root will then be sterilized in an autoclave for 15mins at 1210C and 15 lb pressure. A suspension of Candida albicans will be adjusted to 0.5 turbidity on the McFarland scale
(1.5x108 bacteria/mL). The canal will be then inoculated with 0.5mL of freshly prepared suspension and stored in a glass test tube.
All the samples then will be stored and incubated at 370 C and 91% humidity for 96 hours. Every 24 hours the vials containing experimental teeth will be replenished with freshly prepared suspension. At 48 hours, aliquots will be taken from each tooth using a syringe and will be plated on 4% Sabouraud dextrose agar plate to verify the growth of candida albicans. At the end of 96 hours, all the teeth will be removed from the glass test tube vials and excess fluid in the canal will be removed with sterile paper points.
All the teeth will be randomly divided into three experimental and two control (positive and negative) groups comprising total of one hundred eighteen teeth i.e., thirty six teeth each in experimental group and five teeth each in positive and negative control group.
Standardization of study groups:
Irrigant Group I - 1A – 3% Sodium hypochlorite
1B – 2% Chlorhexidine gluconate
1C – 17% Ethylenediamine tetraacetic acid
Irrigant Group II- 2A – 3% Sodium hypochlorite + Clotrimazole
2B – 2% Chlorhexidine gluconate + Clotrimazole
2C – 17% Ethylenediamine tetraacetic acid + Clotrimazole
Irrigant Group III- 3A – 3% Sodium hypochlorite + Nystatin
3B – 2% Chlorhexidine gluconate + Nystatin
3C - 17% Ethylenediamine tetraacetic acid + Nystatin
Positive control Group IV - Sterile Saline solution.
Negative control Group V- Sterilized samples will be directly irrigated with saline solution.
Samples in the irrigant group I (without antifungal agent) will be further divided into three subgroups consisting of 12 teeth in each group. The experimental teeth in the subgroups then will be rinsed as follows:
1A: 2mL of 3% NaOCl, 1B: 2mL of 2% Chlorhexidine gluconate, and 1C: 2mL of 17% EDTA. The time of contact of each irrigant will be 1min and a final flush of 5mL of distilled water will be performed to terminate the action of the irrigant.
The samples in Group II (with first antifungal agent) will be divided into three subgroups, each containing 12 teeth. The protocol followed for irrigation will be similar to Group I, then followed by irrigation with 2mL of 1% Clotrimazole (10mg/mL), an antifungal irrigant for 1min followed by final flush with 5mL of distilled water.
The samples in Group III, (with second antifungal agent) will be divided into three subgroups, each containing 12 teeth. The initial protocol followed for the irrigation will be similar to the Group I followed by further irrigation with 2mL of 1.5mg/mL of Nystatin for 1min and final flush with 5mL of distilled water.
The positive control Group IV will have five samples and will be irrigated with 1ml of sterile saline solution. The samples in the negative control Group V will have five sterilized samples (inoculation will not be done), and will be irrigated with sterile saline.
Test Method
All experimental teeth will be flushed with 15mL of sterile saline, and canals will be dried with sterile absorbent paper points. A small amount saline solution will be introduced into the canal, and an endodontic hand file will be used in a filing motion. An inoculation loop of 1µm will be used to remove aliquots from the fluid and will be plated on 4% Sabouraud dextrose agar. The plates will be incubated at 360 C and 91% humidity for 48 hours. Then growth of candida albicans will be assessed with light microscope at 400X. The number of colony forming units (CFU) of candida albicans will serve as a measure of antifungal activity. Data will be statistically analysed using appropriate tests.
7.3 Does the study require any investigation or interventions to be conducted on patients or other humans or animals , if so please describe?
-No-
7.4 Has ethical clearance been obtained from your institution in case of 7.3 ?
-Not applicable
8. /
List of references:
1. J. Craig Baumgartner,DDS,PhD, Chad M.Watts, BS, and Tian xia,DDS. Occurrence of Candida albicans in infections of endodontic origin. J Endod 2000 Dec;26(12): 695-8.2. Saurabh S. Chandra, BDS, MDS, Revati Miglani, MDS, DNB, M. R. Srinivasan, MDS, and Rajamani Indira, MDS. Antifungal efficacy of 5.25% sodium hypochlorite, 2%chlorhexidine gluconate, and 17% EDTA with and without an antifungal agent. J Endod 2010 Apr;36(4):675-8.
3. H. Ayhan, N. Sultan, M. Cirak, M. Z. Rsuhi and H. Bodur. Antimicrobial effects of various endodontic irrigants on selected microorganisms. Int. Endo Journal 1999; 32:99-102.
4. Melissa L. Ruff, DMD, MS, Scott B. McClanahan, DDS, MS, and Britta S. Babel, BSMT. In vitro antifungal efficacy of four irrigants as a final rinse.J Endod 2006 Apr;32(4):331-3.
5. Mohmammad Asna Ashari DDS, MS, Fariba Fayaz DDS, MS, Nahid Moezzi Ghadim DDS, Laleh Alim Marvasti DDS, and Yadollah Mehrabi DDS, MS. Evaluation of the antimicrobial effects of MTAD, NaOCl against selected endodontic pathogens. Int. Endodo Journal, Spring 2009;4(2):63-68.
6. Bilge Hakan Sen, DDS, PhD, Kamran E. Safavi, DMD, MEd, and Larz S. W. Spangberg, DDS, PhD. The antifungal effects of sodium hypochlorite and chlorhexidine in root canals. J Endod 1999 Apr;25(4):235-8.