Evaluation of Antimicrobial Efficacy of various Intracanal Medicaments in Primary Teeth: An in vivo Study

Introduction Bacteria and their products play a primary etiological role in the initiation and perpetuation of pulpoperiapical pathosis. Intracanal medication is important for endodontic success as it eliminates microorganisms that persist after chemomechanical preparation. Aim To compare antimicrobial efficacy of calcium hydroxide powder, triple antibiotic paste, calcium hydroxide with 2% chlorhexidine solution, and triple antibiotic paste with 2% chlorhexidine solution. Materials and methods A total of 48 nonvital primary teeth were included in this study. After access opening first microbiological sample (s1) was collected by using absorbent paper point introducing into canal. Second microbilogical sample (s2) was taken following chemomechanical preparation and the teeth were divided into four groups: Group I: calcium hydroxide (CH) powder with distilled water; group II: CH with 2% chlorhexidine solution; group III: triple antibiotic powder with distilled water; group IV: triple antibiotic paste with 2% chlorhexidine solution. Then the canals were filled with any one group of the medicament and cavity was temporarily sealed with zinc oxide eugenol. After 1 week, a postmedication sample (s3) was collected. Then the canal was filled with Metapex, restored with glass ionomer cement. Conclusion From the experiments carried out in this study, with the limitations, an inference can be drawn that a combination of antimicrobial agent used as intracanal medicament is definitely better than single agent like Ca(OH)2. How to cite this article Dutta B, Dhull KS, Das D, Samir PV, Verma RK, Singh N. Evaluation of Antimicrobial Efficacy of various Intracanal Medicaments in Primary Teeth: An in vivo Study. Int J Clin Pediatr Dent 2017;10(3):267-271.


INTRODUCTION
Dental caries, a progressive disease of the dental hard tissue, if left untreated in its initial stages, may lead to pulpal, periapical, and/or intraradicular infections. There is a similarity in the pathogens found in primary teeth to those in permanent teeth. 1 Reduction or elimi nation of the bacterial infection seems to be one of the important factors for the success of endodontic treatment of teeth with necrotic pulp. 2 Although chemomechani cal preparation has an important cleaning effect, it does not ensure the complete elimination of microorganisms present in the root canal system of primary teeth owing to their peculiar internal anatomy and the limitations of the instrumentation technique. 3,4 The remaining viable bacteria left in the root canal may multiply between appointments or following obturation often reaching a pathogenic level and leading to failure. 5 Hence, a suit able medicament is required to disinfect the root canal system prior to obturation so as to promote healing and prevent recurrence. 6 Calcium hydroxide (CH) is commonly used in endo dontics as the intracanal medicament in teeth with necrotic pulp. Its high alkaline pH creates a bacteriostatic environment and the antimicrobial activity is related to its ionic dissociation into calcium and hydroxyl ions and their toxic effects on bacteria, which acts by inhibit ing cytoplasmic membrane enzymes with consequent changes in the organic components and in nutrient transport. 7,8 However, CH is reported to be not much effective against few bacterial strains, especially Enterococcus faecalis. 9,10 Therefore, a combination of agents has been proposed to increase the effectiveness of intracanal medicaments against resistant pathogens. Several other medicaments like chlorhexidine 2% (CHX), either alone or in combination with CH, tripple antibiotic paste (TAP) have been experimented in primary necrotic pulp canals with different degree of success. 1114 However, there is a paucity of literature related to efficacy of various combi nation medication used in primary teeth.
The present study evaluated the antimicrobial effi cacy of a new combination medicament (TAP + CH) and compared with other combinations or CH alone against E. faecalis in necrotic primary teeth in in vivo conditions.

MATERIAlS AND METhODS
Research protocol was approved by the ethical committee of Kalinga Institute of Dental Sciences, KIIT University, Bhubaneswar, Odisha, India, and a written consent was taken prior to the procedure from the parents of children who agreed to participate in the study. Fortyeight chil dren (48 teeth), aged 4 to 6 years, who had open carious lesions in their maxillary or mandibular primary molars with exposure of pulp in clinical diagnosis and radio graphically revealed interradicular/periapical radiolucen cies indicating a necrotic pulp and did not have a history of taking any antibiotics for last 3 months, were included in the study. Children were randomly divided into four groups (three positive control and one experimental) depending on the type of intracanal medicament used: • Group I: CH powder with distilled water • Group II: CH powder with 2% CHX gluconate solution • Group III: Triple antibiotic powder with distilled water • Group IV: Triple antibiotic powder with 2% CHX gluconate solution

Collection of Microbiological Samples
Standard endodontic procedure was performed under rubber dam isolation. Following access cavity prepara tion, orifices of the larger sized canal (distal in mandibular molars and palatal in maxillary molars) were widened using SX hand files (Dentsply) to fascilate easy entry of paper points deep into the canals. First microbiological samples (S1) were collected by inserting #20 (2%) sterile absorbable paper points, leaving it for about 30 seconds and then transfering into a test tube containing brain heart infusion broth. Second samples (S2) were col lected at the end of chemomechanical preparation with SS Kfile (Mani) and 2.5% NaOCl and normal saline. Thereafter, one of the abovementioned medicaments was placed into the root canals using a # 15 Kfile as per the allocated group, and the cavity was temporarily sealed with reinforced ZnOE (Kalsinol, DPI, India). Seven days later, canals were reopened, irrigated with normal saline to wash out the medicaments, and third bacteriological samples (S3) were collected. Canals were then obturated with Vitapex and in subsequent appointments restored with glass ionomer cement (GC Fuji IX, Japan) followed by cementation of SS crown (Kids crown, India).

Microbiological Procedures
Test tubes containing microbiological samples were pre incubated for 30 minutes at 37°C and shaken vigorously in a vertex mixer for 60 seconds. Serial 10fold dilutions were made up to 1:10 6 in 1% sterile saline. From the serial dilutions, 0.1 mL was transferred on to blood agar and MacConkey agar plates. The plates were incubated for 24 hours. All plates were counted for colonyforming units (CFU)/mL after 24 hours. The organisms (E. faecalis) were identified using Gram staining, catalase production, colony morphology on blood agar by using bile esculin agar. Statistical analysis was carried out using Statisti cal Package for the Social Sciences version 20 software program (SPSS Inc, Chicago, IL, USA) and using oneway analysis of variance and Turkey's honest significant dif ference and ttests.

RESUlTS
Out of 48 necrotic primary teeth studied, E. faecalis was isolated from 37 teeth (75%) and results were evaluated only for those 37 teeth.
Enterococcus faecalis counts obtained in the necrosed root canals prior to instrumentation (S1) between the groups were comparable and nonsignificant (p > 0.05) ( Table 1 and Fig. 1). In the second microbiological samples (S2), which was taken immediately after chemomechani cal preparation, a statistically significant reduction in E. faecalis count (p < 0.001) was observed in all groups (Fig. 2). However, while those values were compared between the groups, these differences were not significant. All the four groups exhibited a significant reduction in E. faecalis count (p < 0.001) in third samples (S3) collected 7 days following intracanal medication, compared with their respective values in S2 (Table 2 and Fig. 3). Maximum bacterial inhibition was observed in experimental group (group IV) and minimum in CH group (group I). Intra group comparison at S3 level (Table 2) revealed a highly significant difference in reduction of E. faecalis count between groups I and II, groups I and III, and groups I and IV (p < 0.001). However, the effect of intracanal medi cament in reducing E. faecalis count was not significant between groups II and III and groups II and IV.

DISCUSSION
It should be perceived that majority of teeth indicated for pulp therapy are irreversibly inflammed and only a small percentage of cases exhibit infected or necrotic pulp. The rationale of using intracanal medicament in primary teeth with infected canals is based on the concept that ribbon shaped pulp spaces cannot be completely disinfected following chemomechanical preparation. With the use of intracanal medicaments prior to obturation of root canals, the predictability and prognosis of endodontic success could be enhanced by many fold. Enterococcus faecalis is chosen as test species because of its isolation in therapy resistant apical infection. Besides, in vitro studies have shown its penetration into deeper dentinal tubules 4,15 which can be implicated for recurrence of infection.

IJCPD
The first reported use of antibiotics as an intracanal medicament could be referred to 1951, when Grossman introduced a polyantibiotic paste known as PBSC. 15,16 Thereafter, medicaments like Ca(OH) 2 , CHX (2%), triple antibiotic paste, and propolis have been experimented with varied degree of success. 14,1618 However, with the change in the ecosystem of infected root canals, and prob able mutation of the existing bacterial gene, it is unlikely that a single antimicrobial agent could result in effective sterilization of the canals. Therefore, a combination of

Serial no
The mean (SD) of E. faecalis count (CFU/mL) before instrumentation (S1)
In the present study, a new combination (TAP + CHX) was used to assess its efficacy on E. faecalis count inhibi tion and was compared with CH alone or combination of agents, which have already been experimented. As anticipated, all the four groups displayed statistically significant reduction in bacterial count between S1 and S2 and S2 and S3, confirming the effectiveness of chemome chanical preparation as well as intracanal medicaments in reducing E. faecalis counts. Following intracanal medica ment at S3 level , combination of agents (groups II and III) exhibited better effectiveness in reducing E. faecalis count than CH alone (group I) (p < 0.001). The rationale of using combination of antimicrobial agents to achieve synerg estic effect has also been corroborated by the results of other studies. 2023 Contrary to this, while comparison was made between groups II, III, and IV, group IV displayed maximum inhibition against E. faecalis count than groups III and 4. However, none of the group was significantly better efficacious than the other groups (p > 0.05). Never theless, some in vitro studies demonstrated that CHX gel alone has a greater antimicrobial effect than the combination. 17,19,2427 For any intracanal medicament, to exhibit its biological effect either alone or in combination, several factors like drug concentration, drug interaction, better compaction into the canals, and of course the virulence of the intracanal microorganisms should be relied on. Besides, a difference in individual technique should not also be ignored. Although our study did not reveal that combination of triple antibi otic powder with 2% CHX is not better than other combi nations, further studies with similar type of experiment are required in future to support or oppose our findings.

CONClUSION
From the experiment carried out in this study, with the limitations, an inference can be drawn that a combination of antimicrobial agents used as intracanal medicament is definitely better than a single agent like Ca(OH) 2 . Any combinations commonly used is equally effective against E. faecalis, a resistant intracanal bacteria commonly seen in an infected or necrotic primary root canals.