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ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 304-310
 

Inter-comparison of antimicrobial photodynamic therapy, LASER, and an antifungal agent as adjunct intracanal irrigation techniques to standard disinfection protocols in reducing Candida albicans counts in the root canals of primary teeth: A pilot study


1 Department of Pediatric and Preventive Dentistry, ITS-CDSR, Ghaziabad, Uttar Pradesh, India
2 Oral Pathology, ITS-CDSR, Ghaziabad, Uttar Pradesh, India

Date of Submission20-Jun-2020
Date of Decision11-Aug-2020
Date of Acceptance09-Sep-2020
Date of Web Publication29-Sep-2020

Correspondence Address:
Dr. Pallavi Anand
ITS-CDSR, Muradnagar, Ghaziabad - 201 206, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_286_20

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   Abstract 


Background: Despite endodontic infections being a common problem in the primary dentition, some of the infected primary teeth can remain functional until the exfoliation through endodontic treatment. The primary endodontic treatment goal must be to optimize root canal disinfection and to prevent reinfection. Aim: The aim of this study is to compare the antifungal efficacy of antimicrobial photodynamic therapy, LASER, and 1% clotrimazole as irrigants by reducing the colony-forming units (CFU) of Candida albicans in the infected root canals of primary teeth. Materials and Methods: Pulp therapy was performed on deciduous mandibular second molars of 20 healthy children aged 5–8 years, which were divided into four groups of intracanal disinfection protocols: Group 1 (n = 5) – Sodium Hypochlorite, Group 2 (n = 5) – Photodynamic, Group 3 (n = 5) – Normal saline with LASER, and Group 4 – (n = 5) Antifungal. Disinfection potential was compared by collecting samples one just after access opening and the other after the experimental groups using paper points. The samples were cultured and incubated for 48 h to check change in CFU of the fungi. Results: Data were analyzed statistically using the Shapiro–Wilk's test, Mann–Whitney U-test; Kruskal–Wallis test, and Wilcoxon signed-ranks test. No significant difference (P > 0.05) was found between the different groups. However, complete inhibition of C. albicans CFU was seen with Group 4. Conclusion: One percent Clotrimazole (antifungal) can be utilized as an adjunct in the endodontic irrigation protocol for better success of pulpectomy in children.


Keywords: Antifungal, Candida albicans, diode LASER, root canal disinfection, sodium hypochlorite


How to cite this article:
Anand P, Mathur S, Sachdev V, Jain A. Inter-comparison of antimicrobial photodynamic therapy, LASER, and an antifungal agent as adjunct intracanal irrigation techniques to standard disinfection protocols in reducing Candida albicans counts in the root canals of primary teeth: A pilot study. J Indian Soc Pedod Prev Dent 2020;38:304-10

How to cite this URL:
Anand P, Mathur S, Sachdev V, Jain A. Inter-comparison of antimicrobial photodynamic therapy, LASER, and an antifungal agent as adjunct intracanal irrigation techniques to standard disinfection protocols in reducing Candida albicans counts in the root canals of primary teeth: A pilot study. J Indian Soc Pedod Prev Dent [serial online] 2020 [cited 2020 Oct 22];38:304-10. Available from: https://www.jisppd.com/text.asp?2020/38/3/304/296631





   Introduction Top


Despite the progress made in preventive dentistry, dental caries and its sequelae are among the foremost problems affecting the standard of life in children.[1] The American Academy of Pediatric Dentistry indicated pulpectomy in a primary tooth with irreversible pulpitis within which the radicular pulp exhibits the clinical signs of pulp necrosis or shows evidence of chronic inflammation.[2] Although it has been proven that bacteria causes pulpal disease, numerous studies have revealed a possible role of fungi and more recently viruses in the incidence of endodontic infections.[3]

The incidence of fungi has been reported to be 7%–55% within the infected root canals, Candida albicans being the most common.[4] Sen et al. suggested that Candida could be a “dentinophilic” microorganism as it has collagenolytic activity that makes use of dentin as a nutrient source.[5] In addition to its invading ability, it is also resistant to antimicrobial agents which explain the association of fungal species with retreatment cases.[6]

The success of pulpectomy depends upon optimal irrigation and effective disinfection of the root canals. Conventionally, root canal disinfection has been accomplished by biomechanical preparation and irrigation using disinfecting solutions such as normal saline, chlorhexidine, ethylenediaminetetraacetic acid (EDTA), and sodium hypochlorite.

Newer disinfection methods for infected root canals found to be effective even in tortuous canals like the use of low level laser therapy (LLLT) and antimicrobial photodynamic therapy (aPDT) for root canal disinfection have recently gained attention.

aPDT has been proposed as a new adjunctive method for additional disinfection of the root canal system with the possibility of improved treatment outcomes. It can be defined as “a method of disinfecting a hard tissue or soft-tissue site by topically applying a photosensitizer to the site, and then irradiating it with LASER at a wavelength absorbed by the photosensitizing compound, to destroy microbes at the site.“[7]

Furthermore, some antibiotics pastes containing antifungals (like Nystatin and Sodium caprylate) were extensively used both locally and systemically for sterilizing infected root canals during the 1960s and 1970s.[8]

Thus, the purpose of this study is to compare the efficacy of aPDT, LASER, and 1% Clotrimazole as adjunct intracanal irrigation techniques to standard disinfection protocol by reducing the colony-forming units (CFU) of C. albicans in the infected root canals of primary teeth. The null hypothesis was that the ability to eliminate C. albicans would not vary significantly among the various root canal disinfection techniques.


   Materials and Methods Top


Prior ethical approval was taken by the Internal Ethical Committee, and informed consent was obtained from each subject's parents/guardians before enrolling them in this study. This pilot study was conducted among a sample size of 20 patients calculated using the Statistical Package for the Social Sciences (SPSS) software package (SPSS 16 Inc., Chicago, IL, USA) for this study according to the following formula:

Sample size





S1= Standard deviation of Group A, S2= Standard deviation of Group B, μd2 = Mean difference between the samples, α = Level of significance, and 1−β = Power.

Healthy patients aged 5–8 years who were indicated for pulpectomy procedures in deciduous mandibular second molar teeth were included this study. However, teeth with the presence of large periapical lesion, internal or external root resorption, and medically compromised patients were excluded from the study.

After administering local anesthesia, the tooth requiring pulpectomy was isolated using the rubber dam. Immediately, after the access opening was done [Figure 1], first sample (S1) was collected from the canal using sterile no. 15 paper point [Figure 2] and was transferred to 2 ml of freshly prepared glucose broth, and also, the solution was agitated [Figure 3].
Figure 1: Access opening of teeth

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Figure 2: Collection of sample using sterile paper point

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Figure 3: Transfer of sample in 2 ml glucose broth

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The working length was determined 0.5 mm short of the anatomic apex with the help of digital radiograph using number 10 K-file (Dentsply Maillefer, OK, USA). The canals were further prepared with hand instrumentation using hand K-file up to number 35 K-file using quarter turn and pull motion. In-between subsequent filing, the root canals were copiously irrigated with sterile saline solution. After completion of cleaning and shaping, so as to eliminate the smear layer, the canals were irrigated with 2 ml of 17% EDTA for 1 min followed by disinfection with 2 ml of 2.5% sodium hypochlorite for 1 min.[9]

Study design

After this standard irrigation protocol; the participants were randomly divided into 4 groups:

  • Group 1-Sodium Hypochlorite group: A second sample (S2) was collected from the canals using sterile 30 number paper point and transferred to glucose broth without intervention
  • Group 2 Photodynamic therapy group: 0.5 ml Methylene blue dye was injected into the coronal part of the tooth. The activated LASER tip was used at 0.2-watt power in continuous noncontact mode and 200μ diameter fiber was inserted in the coronal part and moved in circular motion for three cycles (10 seconds each); S2 was collected
  • Group 3-Normal saline with LASER group: Normal saline was incorporated in the canals and LLLT with 940 nm Diode LASER (ilase, Biolase Technology Inc., U. S. A) using 200-micron diameter fiber tip at 0.5-Watt power in continuous noncontact mode was delivered inside each root canal from the apical area till the canal opening moving it in a retrieving circular motion at the rate of 1 mm/s. Three cycles of irradiation were done per the root canal. S2 was collected in the same way as mentioned before
  • Group 4-Antifungal group: Irrigation with 2 ml of 1% Clotrimazole (Candid Mouth paint, Glenmark, India) was done before collecting S2.


Finally, the canals were flushed with 5 mL of saline to remove any debris and residual irrigants. All the teeth were obturated using Endoflas.

Fungal evaluation

Samples were inoculated on Sabouraud Dextrose Agar (Himedia, India) [Figure 4] a selective medium for Candidia albicans and incubated at 37°C at 91% humidity for 48 h [Figure 5] Colony counting was done using digital colony counter. The number of CFU of Candida served as a measure of antifungal activity.[9]
Figure 4: Sabaouraud dextrose agar for cultivation of Candida albicans

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Figure 5: Candida albicans count on sabaouraud dextrose agar-(a) Preoperative count in group 1; (b) Postoperative count in group 1; (c) Preoperative count in group 2; (d) Postoperative count in group 2; (e) Preoperative count in group 3; (f) Postoperative count in group 3; (g) Preoperative count in group 4; (h) Postoperative count in group 4

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Statistical analysis

Statistical tests were performed using the SPSS software (SPSS 16 Inc., Chicago, IL, USA). Data were analyzed statistically using the Shapiro-Wilk's test, Mann–Whitney U-test, Kruskal-Wallis test, and Wilcoxon signed-ranks test. The level of statistical significance was set at P < 0.05.


   Results Top


The parameters studied for various root canal irrigation protocols were the Pre and Postoperative CFU of C. albicans. The mean preoperative and postoperative fungal CFU values are shown in [Table 1] and graphically depicted in [Graph 1]. [Table 2] show preoperative CFU values of pairs of four groups (P > 0.05) depicting that baseline CFU of Candida samples were uniformly distributed between all 4 groups. Similarly, postoperative CFU values in [Table 3] shows no significant difference (P > 0.05) which proves that all the protocols used for disinfection of root canals of primary teeth in this study were equally effective. Krushal Wallis test check the difference between Pre and Post CFU values for all four groups. No significant difference in pre- and post-values was found when all groups were compared (P = 0.583 > 0.05) in [Table 4].
Table 1: The mean Pre-operative and Postoperative fungal CFU values

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Table 2: Pre-operative CFU values of pairs of four groups for intergroup comparison using Mann-Whitney U Test

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Table 3: Post-operative CFU values of pairs of four groups for intergroup comparison using Mann-Whitney U Test

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Table 4: Comparison between Pre-operative and Post-operative CFU values for all four groups

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   Discussion Top


Literature supports that the success rate of a negative microbiological culture of a root canal before obturation is as high as 94%.[10] The anatomical complexity of the deciduous root canal system with its isthmuses, ramifications, make complete debridement of microbes almost impossible, even when conventional methods of endodontic instrumentation, and irrigation are performed to the highest technical standards.[11] Endodontic infections are polymicrobial, aside from gram-positive cocci and obligate anaerobes, fungi also have been isolated from the root canals of infected teeth. Among the fungi, C. albicans is the most common and the most resistant to endodontic procedures and showed an ability to colonize the canal walls and invade dentinal tubules.[12]C. albicans was taken as the test organism in this study due to its presence in 7%–18% cases of persistent apical periodontitis, 30%–45% of post treatment diseases and due to high level of resistance to a wide range of antimicrobial agents.

According to Waltimo et al., sodium hypochlorite showed the best efficacy against C. albicans in therapeutic concentrations. This was also supported by Lau H et al. proving that 2.5% sodium hypochlorite used for 1 min in infected root canals of primary teeth can eliminate C. albicans.[13] Goztas Z et al.[14] concluded sodium hypochlorite irrigation was found effective in primary molar root canal which was in accordance with Baumgartner and Mader[15] who confirmed that 2.5% sodium hypochlorite is extremely effective in removing vital pulp tissue from dentinal walls.

Although during endodontic treatment, irrigation with sodium hypochlorite which is considered as the gold standard and removes the majority of infecting microorganisms, it is still possible to regain infection of the root canal and some cases have shown that occasionally C. albicans has been associated with endodontic failures.

We did not find manyin vivo studies proving the effectiveness of aPDT in disinfecting the infected root canals of primary teeth. Souza RCet al. conducted anin vitro study which concluded that both aPDT and low level laser irradiation had a fungicidal effect on C. albicans.[16]

In recent years, aPDT has been suggesting its usefulness as an adjunct to current endodontic disinfection techniques. It relies on the topical or systemic administration of a nontoxic photosynthesizer (PS), followed by low dose irradiation with visible light of a suitable wavelength. Absorption of the light triggers excitation of the PS which, in the presence of oxygen, results in a cascade of photochemical effects, leading to the production of high reactive oxygen species that are toxic to tumor cells, bacteria, and fungi.[17] The largest amount of oxygen is generated from 0.01% Methylene Blue (MB) and is increased by laser irradiation in a dose-dependent manner.[18] MB which is Food and Drug Administration approved was used as the photosensitizer in this study because of its superior hydrophilicity along with its low molecular weight.

However, some studies suggested that all microbial strains tested seemed to be sensitive to photodynamic therapy, at some level with exception of C. albicans.[17] This could be because the photosensitizer agent might not diffuse well into the dentinal tubules and may not achieve direct contact with the fungi on the root canal walls, especially in tubules and irregularities, when compared with other irrigants.

For our study, we used a 940 nm wavelength Diode LASER Epic 10 (Biolase, Inc.) which is known as a tissue-friendly LASER showing lesser temperature rise compared with other LASER, like Nd-YAG.[1]

In our study, interestingly, Group 4 showed complete inhibition of posttreatment counts of C. albicans in primary root canals. Clotrimazole, a substituted imidazole, is a commonly used antifungal in both medical and dental practice. The imidazoles are preferred over other antifungal drugs due to their greater efficacy, fewer side effects, and lesser drug interactions and their broad-spectrum antifungal activity covering the Candida species, dermatophytes, and a few Gram positive and anaerobic bacteria like Staphylococcus aureus and Streptococcus faecalis. The mechanism of action is by inhibition of the fungal cytochrome P450 enzyme to block demethylation of lanosterol 14-demethylase, which impairs the ergosterol synthesis resulting in a cascade of membrane abnormalities in the fungus. This inhibition disrupts membrane function and increases permeability.[19] For this study, we chose 1% clotrimazole mouth paint, which is regularly used for the treatment of oral candidiasis and is a cost-effective disinfection method to be used as an adjunct to the standard irrigation protocols of endodontic treatment in primary teeth.

Under the parameters of the present study, sodium hypochlorite, aPDT, diode LASER, and Clotrimazole disinfection protocols for infected root canals of primary teeth were found to be equally effective with statistically no significant difference (P > 0.05) in the postoperative CFU of C. albicans. The effectiveness of sodium hypochlorite on C. albicans is well documented in literature.[5],[8] Wadhwani et al. concluded that 5.25% sodium hypochlorite exhibited superior antifungal efficacy compared to 2% chlorhexidine gluconate and 17% EDTA for root canal disinfection. However, on inclusion of 1% clotrimazole, 5.25% sodium hypochlorite and 2% chlorhexidine gluconate showed significantly greater antifungal properties than 17% EDTA with clotrimazole.[3] Babu B et al. even concluded that clotrimazole showed superior efficacy than fluconazole in the inhibition of growth of C. albicans when used in conjunction with standard irrigation protocol.[9]

Although the incidence of Candida is 1%–17% in infected root canals, higher titers are observed in patients with AIDS or other immunocompromised states. A poorly functioning immune system might increase the risk of fungal infection in the root canal system.[20] Therefore, disinfection of the root canal during cleaning and shaping procedures included with an effective antifungal agent is of great importance not just in healthy individuals but also in immunocompromised patients which can have a history of oral candidiasis, to target the higher number of CFU of fungi specifically. Each irrigant has a specific action and must be used in the combination of other irrigants in clinical practice to make sure complete disinfection of root canal space.

Evaluating antimicrobial activity against only one organism represents a limitation to the current study, since the presence of multiple microorganisms might have altered the dynamics demonstrated by the present study. Molecular techniques such as polymerase chain reaction are more sensitive than culture methods. Higher number of microbial species has been identified using molecular detection methods.[21] However, molecular techniques cannot completely replace the culturing methods as they cannot give information about the viability of the organism.


   Conclusion Top


  • Under the parameters of the present study, it was concluded that aPDT, diode LASER, and Clotrimazole disinfection protocols for infected root canals of primary teeth are equally effective as an adjunct to standard irrigation protocol with no significant difference (P > 0.05) in the postoperative CFU of C. albicans
  • This study throws light on the antifungal efficacy of LLLT and aPDT as intracanal irrigation techniques in the infected root canals of primary teeth
  • The results of this study also indicate that 1% Clotrimazole showed complete inhibition of C. albicans CFU in the root canal space of pediatric patients.


Hence, our findings suggest the utilization of 1% Clotrimazole as a cost-effective adjunct to the standard endodontic irrigation protocol for better success of pulpectomy in children.

Further, extensive studies on large number of samples are required to establish more reliable results forming a conclusive recommendation to intracanal disinfection protocols in the infected root canals of primary teeth.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Öter B, Topçuog Lu N, Tank MK, Çehreli SB. Evaluation of antibacterial efficiency of different root canal disinfection techniques in primary Teeth. Photomed Laser Surg 2018;36:179-84.  Back to cited text no. 1
    
2.
American Academy of Pediatric Dentistry. Pulp therapy for primary and immature permanent teeth. Ref Manual Pediat Dent 2014;40:343-51.  Back to cited text no. 2
    
3.
Chandra SS, Miglani R, Srivasan MR, Indira R. Antifungal efficacy of 5.25% sodium hypochlorite, 2% chlorhexidine gluconate, 17% EDTA with or without as antifungal agent. J Endod 2010;36:675-78.  Back to cited text no. 3
    
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Jackson FL, Halder AR. Incidence of yeasts in root canals during therapy. Brit Dent J 1963;115:459-60.  Back to cited text no. 4
    
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Sen BH, Safavi KE, Spangberg LS. Growth patterns of Candida albicans in relation to radicular dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997;84:68-73.  Back to cited text no. 5
    
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Siqueira JF Jr., Rôças IN, Lopes HP, Lopes HP, Magalhães FA, de Uzeda M. Elimination of Candida albicans infection of the radicular dentine by intracanal medicaments. J Endod 2003;29:501-04.  Back to cited text no. 6
    
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Ahmad MZ. Anti-microbial photo dynamic therapy (aPDT) in endodontics: A literature review. Int J Dent Sci Res 2018;6:25-8.  Back to cited text no. 7
    
8.
Sen BH, Safavi KE, Spångberg LS. Antifungal effects of sodium hypochlorite and chlorhexidine in root canals. J Endod 1999;25:235-8.  Back to cited text no. 8
    
9.
Babu B, Nair RS, Angelo JM, Mathai V, Vineet RV. Evaluation of efficacy of chitosan-silver nanocomposite on Candida albicans when compared to three different antifungal agents in combination with standard irrigation protocol: An ex vivo study. Saudi Endo J 2017;7:87-91.  Back to cited text no. 9
    
10.
Nair PN, Sjögren U, Krey G, Kahnberg KE, Sundqvist G. Intraradicular bacteria and fungi in root-filled, asymptomatic human teeth with therapy-resistant periapical lesions: A long-term light and electron microscopic follow-up study. J Endod 1990;16:580-8.  Back to cited text no. 10
    
11.
Siqueira JF Jr., Rocas IN, Paiva SS, Magalhães KM, Pinto TG. Cultivable bacteria in infected root canals as identified by 16S rRNA gene sequencing. Oral Microbiol Immunol 2007;22:266-71.  Back to cited text no. 11
    
12.
Waltimo TM, Sen BH, Meurman JH, Ørstavik D, Haapasalo MP. Yeasts in apical periodontitis. Crit Rev Oral Biol Med 2003;14:128-37.  Back to cited text no. 12
    
13.
Lau H, Ballal V, Shenoy S, et al. Evaluation of antifungal efficacy of 5% doxycycline hydrochloride,2.5% sodium hypochlorite, 17% ethylenediamine tetraacetic acid and 0.2% chlorhexidine gluconate against candida albicans – an in vitro study. Int Endod J 2008; 20:6-13.  Back to cited text no. 13
    
14.
Goztas Z, Onat H, Tosun G, Sener Y, Hadimli HH. Antimicrobial effect of ozonated water, sodium hypochlorite and chlorhexidine gluconate in primary molar root canals. Eur J Dent 2014;8:469-74.  Back to cited text no. 14
  [Full text]  
15.
Baumgartner JC, Mader CL. A scanning electron microscopic evaluation of four root canal irrigation regimens. J Endod 1987;13:147-57.  Back to cited text no. 15
    
16.
Souza RC, Junqueira JC, Rossoni RD, Pereira CA, Munin E, Jorge AO. Comparison of the photodynamic fungicidal efficacy of methylene blue, toluidine blue, malachite green and low power laser irradiation alone against Candida albicans. Lasers Med Sci 2010;25:385-9.  Back to cited text no. 16
    
17.
Trindade AC, De Figueiredo JA, Steier L, Weber JB. Photodynamic therapy in endodontics: a literature review. Photomed Laser Surg. 2015;33(3):175-182.  Back to cited text no. 17
    
18.
Schlafer S, Vaeth M, Hørsted-Bindslev P, Frandsen EV. Endodontic photoactivated disinfection using a conventional light source: Anin vitro and ex vivo study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:634-41.  Back to cited text no. 18
    
19.
Chandra SS, Miglani R, Srinivasan MR, Indira R. Antifungal efficacy of 5.25% sodium hypochlorite, 2% chlorhexidine gluconate, and 17% EDTA with and without an antifungal agent. J Endod. 2010;36(4):675-678.   Back to cited text no. 19
    
20.
Ruff ML, McClanahan SB, Babel BS. In vitro antifungal efficacy of four irrigants as a final rinse. J Endod 2006;32:331-3.  Back to cited text no. 20
    
21.
Munson MA, Pitt-Ford T, Chong B, Weightman A, Wade WG. Molecular and cultural analysis of the microflora associated with endodontic infections. J Dent Res 2002;81:761-6.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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