ENDODONTOLOGY INTRODUCTION. Original Research - 1 ABSTRACT

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1 Original Research - 1 Evaluation of antifungal efficacy of % doxycycline hydrochloride, 2.% sodium hypochlorite, 17% ethylenediamine tetraacetic acid and 0.2% chlorhexidine gluconate against candida albicans - An in vitro study HIMANI LAU * VASUDEV BALLAL ** SHALINI SHENOY *** SHASHI RASHMI ACHARYA **** ABSTRACT The purpose of this study was to evaluate the antifungal efficacy of % doxycycline hydrochloride (Doxy-HCl) and its comparison with the antifungal efficacy of 2.% sodium hypochlorite, 17% EDTA and 0.2% chlorhexidine gluconate against Candida albicans in vitro. Twenty-five, single rooted extracted human teeth were randomly divided into five groups with saline as negative control. After root canal preparation, experimental teeth were inoculated with the standard strain (ATCC 24433) of Candida albicans and incubated for 24 and 72 hours. The specimens were irrigated as follows: 2 ml of 2.% NaOCl, 2 ml of 17% EDTA, 2 ml of 0.2% CHX and 2 ml of % Doxy-HCl for 1 minute. Aliquots from the experimental teeth were transferred on Sabouraud s dextrose agar (SDA) plates with paper points and colonyforming units were counted as a measure of antifungal activity. The results showed that % Doxy-HCl had an antifungal efficacy and was statistically significant (p<0.002) to 17% EDTA and 2.% NaOCl. Key words: Candida albicans, doxycycline hydrochloride, sodium hypochlorite, EDTA, chlorhexidine gluconate INTRODUCTION Micro-organisms cause virtually all pathosis of the pulp and peri-radicular tissues. Polymicrobial interactions and nutritional requirements make the cultivation and identification of all organisms from endodontic infections very difficult. There can be no doubt today that microorganisms, either remaining in the root canal space after treatment or re-colonising the filled canal system are the main cause of endodontic failure. The primary endodontic treatment goal must be to optimise root canal disinfection and to prevent reinfection. 1 Biomechanical preparation and chemical disinfection are used concomitantly to cleanse the root canal system. 2 Chemical disinfection involves thorough flushing of root canal system. 3 Fungi have been detected in infected root canals, but the number of yeast cells in the root canal is usually lower than that of bacteria. 4 * Post-Graduate student, ** Reader, **** Professor and Head, Dept. of Conservative Dentistry and Endodontics, MCODS, Manipal, Karnataka, India. ***Professor and Head, Dept. of Microbiology, KMC, Mangalore, Karnataka, India 6

2 EVALUATION OF ANTIFUNGAL EFFICACY OF % DOXYCYCLINE HYDROCHLORIDE, 2.% SODIUM HYPOCHLORITE, 17% ETHYLENEDIAMINE TETRAACETIC ACID AND 0.2% CHLORHEXIDINE GLUCONATE AGAINST CANDIDA ALBICANS AN IN VITRO STUDY Yeasts, particularly Candida albicans have been isolated from infected dental pulp and root canals. It is the most commonly isolated fungi from oral cavity of both healthy and medically compromised individuals. It is the most infective and invasive yeast among the Candida species. Candida is versatile and can adapt to a range of ph change, gene expression in response to environmental cues, adhere to a variety of surfaces, produce degradative enzymes, and change morphologic forms to evade the immune system. 6 It has collagenolytic activity that makes use of dentin as a nutrient source. 7 It has a particular affinity to dentin and smear layer. 8 It has been demonstrated in root canals of therapy-resistant cases. 9 that invasion of dentinal tubules and root canals can cause periapical granulomas 10 or abscesses. 11 The incidence of yeasts in infected root canals is reported to vary between 7% and %. 12 In addition to its invading ability, it is also resistant to some intracanal medicaments after direct contact which explains why this fungal species has been associated with cases of persistent root canal infections. 13 Sodium hypochlorite (NaOCl) is one of the commonly used irrigant because of its antimicrobial, sporicidal, virucidal, tissue dissolving properties and aids in debridement of canal system. 1 Candida has proved very susceptible to its action in as little as 0.% concentration within 10 seconds of contact time. 14 1% and % concentration has showed antifungal activity in the presence of smear layer only after treatment period of 1 hour. In the absence of smear layer, % concentration demonstrated antifungal activity after 30 minutes. 1 Chlorhexidine gluconate, a broad spectrum antimicrobial, is less toxic than hypochlorite, but it is unable to dissolve necrotic tissue remnants or remove the smear layer and is less effective on gram-negative bacteria. 1 It displays affinity to dentin, forms deposits, and has effective antimicrobial activity. It has been shown that calbicans was resistant to antibacterial effects of calcium hydroxide medication, but was sensitive to chlorhexidine. 16 EDTA, a chelating agent has shown to have the most effective antifungal activity. 1 It has anticolonisation, anti-growth and anti-collagenolytic properties against C.albicans. By chelating calcium ions in the medium, EDTA prevents binding of C.albicans to the proteins in a dose-dependent manner. In the second process, EDTA reduces the growth of C.albicans by removing calcium from the cell walls and causing collapse in the cell wall and by inhibiting enzyme reaction. Using the agar diffusion method, Sen et al demonstrated that 17% EDTA had the highest antifungal activity in comparison with routine antifungal drugs and other test solutions. 17 Doxycycline, a broad spectrum antibiotic and hydroxy derivative of tetracycline, is the most potent anti-collagenase antibiotic among the commercially available tetracyclines. A further benefit of doxycycline is that, prolonged treatment with the drug, does not facilitate bacterial mutation to generate tetracycline-resistant microorganisms. 18 Doxycycline hydrochloride is an effective acidic conditioner (ph=2), which aids in smear layer removal. 19 It readily attaches to dentin and is subsequently released without losing its 7

3 HIMANI LAU, VASUDEV BALLAL, SHALINI SHENOY, SHASHI RASHMI ACHARYA antibacterial property. This property creates a reservoir of active antibacterial effect which is released from dentin surfaces in a slow and sustained manner. It is inexpensive and easily available as tablets in dispersible form. It forms one of the components of MTAD. However, the efficacy of doxycycline hydrochloride on fungi is unknown. The purpose of this study was to evaluate the antifungal efficacy of % Doxy-HCl and its comparison with the antifungal efficacy of 2.% NaOCl, 17% EDTA and 0.2% CHX against Candida albicans in vitro. MATERIALS AND METHOD Preparation of specimens Twenty five extracted single rooted human teeth were used in this study. They were then sectioned horizontally 14 mm from the apex using a carborundum disc at slow speed. Root canal instrumentation was performed by step-back technique using Gates Glidden drills #3 & #2 and K-files. Apical preparation was done upto ISO size 40. Then step back preparation was done upto ISO size 60. Canals were irrigated with 1 ml of.2% sodium hypochlorite between each files. The smear layer was removed with 1 ml of 17% EDTA followed by 3 ml of.2% sodium hypochlorite. The canals were flushed copiously with sterile saline to remove the residual irrigants. Apical foramina were sealed with glass ionomer cement (GIC Fuji II) and the teeth were coated with nail varnish. The roots were then kept in small plastic vials, which were sterilised in the autoclave. Inoculation Standard strain of Candida albicans (ATCC 24433) was obtained from the Department of Microbiology, Kasturba Medical College, Mangalore. A suspension of Candida albicans was adjusted to 0. turbidity on the McFarland scale. Vials containing teeth were randomly divided into five groups and each group consisted of five teeth each. A 0.3 ml of the adjusted C. albicans suspension was injected with a sterile syringe into the canal and into the tubes to submerge the teeth. The samples were incubated at 37ºC for 3 days. At every 24 hours, the vials containing the teeth were replenished with freshly made suspensions of C.albicans. At 48 hours, 1 µl suspension was taken from each tooth using a calibrated inoculation loop and plated on Sabouraud s dextrose agar (SDA) plates to verify the growth of C.albicans in each sample tube. Growth was verified with light microscopy at X 400. After verification of growth of C. albicans in root canal system, the final rinse of test solutions was performed at 72 hours. Irrigation Gross excess fluid was removed from the canals with sterile paper points to prevent the dilution of the irrigants before the experiment. The samples were rinsed with 2. ml sterile syringe with the test solutions as follows: GROUP A: 2 ml of 2.% NaOCl solution for 1 minute. GROUP B: 2 ml of 17% EDTA solution for 1 minute. 8

4 EVALUATION OF ANTIFUNGAL EFFICACY OF % DOXYCYCLINE HYDROCHLORIDE, 2.% SODIUM HYPOCHLORITE, 17% ETHYLENEDIAMINE TETRAACETIC ACID AND 0.2% CHLORHEXIDINE GLUCONATE AGAINST CANDIDA ALBICANS AN IN VITRO STUDY GROUP C: 2 ml of 0.2% CHX solution for 1 minute. GROUP D: 2 ml of % Doxy-HCl solution for 1 minute. GROUP E: 2 ml of 0.9% saline for 1 minute (negative control). All experimental teeth were then flushed with 30 ml distilled water to prevent potential carry over of the irrigants. SAMPLING TECHNIQUE Sterile paper points were used to collect the samples from the root canals. It was then cultured on Sabouraud s dextrose agar plates, which were incubated at 37ºC for 24 hours. After incubation period, the number of colonies grown were counted and multiplied with the dilution factor (1000) to calculate the colony forming units (CFUs) per ml and their average was tabulated. All procedures were carried out under aseptic conditions. The CFU count was taken after 24 and 72 hrs. Data were analysed using Kruskal-Wallis and Mann-Whitney Rank Sum Test (p < 0.002). RESULTS Table 1 and 2 demonstrates the mean CFUs of Candida albicans after irrigating with the test irrigants after 24 hrs and 72 hrs respectively. 0.2% CHX (p<0.002) showed the best results. 2.% NaOCl was the second best (p<0.002), followed by % Doxy-HCl (p<0.002).and 17% EDTA (p<0.002) respectively which was statistically significant in both 24 and 72 hrs. TABLE 1 CFU 24 HRS N Mean Std. Deviation Minimum Maximum H E C D Saline H= p=0.002 hs TABLE 2 CFU 72 HRS N Mean Std. Deviation Minimum Maximum H E C D Saline H= p=0.002 hs 9

5 COMPARISON OF MEAN ANTIFUN- GAL EFFICACY OF TEST IRRIGANTS DISCUSSION Microorganisms that survive chemomechanical procedures and the application of root canal medicaments are the Enterococci, Streptococci, Lactobacilli, and some fungi such as Candida albicans. 20 SEM analysis showed that Candida albicans builds colonies along the dentin in the root canal and penetrates the dentinal tubules; this defines it as a dentinophile microorganism compared to other species of fungi. Candida albicans is described as a dimorphic and polymorphic fungus. 21 It is well established that cleaning and shaping of the root canal system, using files and antibacterial irrigants, reduces the bacterial load. As part of the debridement procedure besides instrumentation of the root canal, irrigation with an appropriate agent is essential. However, no irrigant can completely eliminate all organic and inorganic matter and at the same time impart a substantive residual antibacterial property to the canal wall dentin. 22 HIMANI LAU, VASUDEV BALLAL, SHALINI SHENOY, SHASHI RASHMI ACHARYA In our study, C. albicans was inoculated and grown on root canal walls to form a biofilm to simulate the intraoral condition. Considering that high concentrations of dietary sugars are often found in the mouth and coaggregation reactions with bacteria favor its growth and colonisation, C. albicans may show more resistance and pathogenicity in vivo. 23 Sodium hypochlorite is a commonly used endodontic irrigant. Regarding its antifungal efficacy, it has been shown to exhibit complete antifungal activity in a range from 1 seconds to minutes. 24 In the current study, the results show that 2.% sodium hypochlorite is very effective against Candida albicans in a time period of one minute. Chlorhexidine gluconate is a strong antiseptic. Since a higher concentration can be toxic, we used 0.2% concentration in our study, which demonstrated the highest antifungal activity. The results of our study proves the antifungal action of chlorhexidine which is found to be higher than sodium hypochlorite which is statistically significant. This result is in accordance with the previous studies which have shown that 0.12% chlorhexidine possesses antifungal properties as an endodontic irrigant. 2 EDTA (ph -7.3) has been widely used as an endodontic irrigant. In this study, 17% EDTA solution demonstrated an antifungal activity. This was in accordance with the previous studies which showed EDTA being used as an alternative irrigant in persistent root canal infections and root canals of patients having high incidence of oral candidosis

6 EVALUATION OF ANTIFUNGAL EFFICACY OF % DOXYCYCLINE HYDROCHLORIDE, 2.% SODIUM HYPOCHLORITE, 17% ETHYLENEDIAMINE TETRAACETIC ACID AND 0.2% CHLORHEXIDINE GLUCONATE AGAINST CANDIDA ALBICANS AN IN VITRO STUDY Doxycycline is a broad spectrum antimicrobial, primarily bacteriostatic, inhibits protein synthesis by binding to 30S bacterial ribosomes in the susceptible organism. Studies have shown that doxycycline hydrochloride removes smear layer, binds to dentin and eliminates E. faecalis in 2 or minutes. The higher doses of doxycycline hydrochloride solution are toxic and inhibit fibroblast adherence. Hence, lower concentration (%) was used in our study. Some studies mention that doxycycline is active against a wide range of gram- positive and gram- negative organisms, but is not active against fungi. 27 Since there are no known reports of the antifungal efficacy of doxycycline hydrochloride and its comparison with the known irrigants, this in-vitro study was an attempt to find out the same. In our study, doxycycline hydrochloride showed a good antifungal activity as compared to EDTA and saline. This could be attributed to its better flow and penetration properties. Studies have suggested that sodium hypochlorite and chlorhexidine digluconate diffuses through the root canal, and possibly into the dentinal tubules; they will be effective candidacidal agents, even if they become significantly diluted in the process. 28 On the contrary, doxycycline hydrochloride was shown to be less effective than sodium hypochlorite and chlorhexidine digluconate because of a lesser penetration ability. The in vitro model used here was adapted from that used by Ørstavik and Haapasalo. This model was adapted for extracted teeth, which reflects the clinical situation better than do bovine incisors. A standard loop of 4mm size was used which carried 0.01 ml of the candidal suspension. Therefore, a dilution factor of 1000 was multiplied by the colony forming unit (CFU) count to give CFU per ml. 30 ml volume of saline was used as final irrigant to eliminate prolonged contact time of each irrigant and to standardise the groups. Therefore, the potential carry over of antifungal activity to the culture medium should be considered. One minute of contact time was chosen in this study because previous studies found that less than 1 minute of contact time was enough to eliminate C.albicans when sodium hypochlorite or chlorhexidine were used. 14 We observed that more than 70% of the samples presented negative C. albicans culture after the use of 2.% sodium hypochlorite solution. The growth of yeast in 30% of the samples may be due to the difficulty of penetration of this irrigant into root canal irregularities. Similar results were obtained by Siqueira et al in The control group, which was irrigated only with 0.9% sterile saline solution, showed C. albicans growth in 90% of the samples. This result was similar to that shown by Valera et al. 30 Ninety-two percent of patients with a diagnosis of AIDS may present a history of oral candidiasis during the course of their disease. Invasion of dental tissues, including root canals by yeasts, will be inevitable with this high percentage of oral candidiasis. Therefore, the selection of an irrigating solution in root canals of medically compromised patients gains extreme importance. The use of specific antifungal agents may be recommended in the endodontic therapy of patients having local or 11

7 HIMANI LAU, VASUDEV BALLAL, SHALINI SHENOY, SHASHI RASHMI ACHARYA systemic predisposing factors to oral candidiasis. One limitation of this study is that a single organism was used to infect the root canal. Because endodontic infections are polymicrobial, interactions between multiple organisms could potentially have different dynamics than were demonstrated by this study. The irrigant that is effective against single microbe in vitro may not necessarily be effective against the same microbe in vivo since the root canal system contains multiple microorganisms. Doxycycline hydrochloride can be used effectively against Candida albicans as a root canal irrigant. It being economically affordable, easily available, will prove to be an effective endodontic irrigant against C.albicans in the years to come. CONCLUSIONS Under the parameters of this study, it was concluded that: 1) % Doxy-HCl showed an antifungal efficacy against Candida albicans as a root canal irrigant. 2) % Doxy-HCl showed better antifungal effect than 17% EDTA. 3) Among the solutions used for irrigating the root canals, 0.2% CHX showed the best antifungal efficacy against C.albicans, followed by 2.% NaOCl, % Doxy-HCl and 17% EDTA. ACKNOWLEDGEMENTS The author would like to thank Dr. M Kundabala for her esteemed guidance and support. References 1. Zehnder M. Root canal irrigants. J Endod 2006;32(): Harrison JW. Irrigation of the root canal system. Dent Clin North Am 1984;28(4): Ingle. Endodontics 200: Waltimo TM, Haapasalo M, Zehnder M, Meyer J. Clinical aspects related to endodontic yeast infections. Endodontic Topics 2004;9: As cited in J Endod 2006;32: Siqueira JF, Rocas IN, Lopes HP, Elias CN, de Uzeda M. Fungal Infection of the radicular dentin. J Endod 2002;28: Calderone RA, Fonzi WA. Virulence factors of Candida albicans. Trends in Microbiol 2001;9: As cited in J Endod 2006;32: Hagihara Y, Kaminishi H, Cho T, Tanaka M, Kaita H. Degradation of human dentine collagen by an enzyme produced by the yeast Candida albicans. Arch Oral Biol 1988;33: Sen BH, Safavi KE, Spangberg LS. Colonisation of Candida albicans on cleaned human dental hard tissues. Arch Oral Biol 1997;42: Waltimo TMT, Siren EK, Torkko HLK, Olsen I, Haapasalo MPP. Fungi in therapy-resistant apical periodontitis. Int Endod J 1997;30: Eidelman D, Neuman I, Kuttin ES, Pinto M, Beemer AM. Dental sepsis due to Candida albicans causing urticaria: case report. Ann Allergy 1978;41: Damm DD, Neville BW, Geissler RH Jr, White DK, Drummond JF, Ferretti GA. Dentinal candidiasis in cancer patients. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1988;6: Jackson FL, Halder AR. Incidence of yeasts in root canals during therapy. Brit Dent J 1963;11: Jose F Siqueira, Isabela N, Rocas et al. Elimination of Candida albicans infection of the radicular dentine by intracanal medicaments. J Endod 2003;29(8): Radcliffe CE, Potouridou L, Qureshi R, et al. Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A.naeslundii, Candida albicans and Enterococcus faecalis. Int Endod J 2004;37: Jeansonne MJ, White RR. A comparison of 2% chlorhexidine gluconate and.2% sodium hypochlorite as antimicrobial endodontic irrigants. J Endod 1994;20: White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod 1997;23: Bilge Hakan Sen, B. Guniz Akdeniz, A. Akin Denizci. The effect of ethylenediamine-tetraacetic acid on Candida albicans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2000;90:

8 EVALUATION OF ANTIFUNGAL EFFICACY OF % DOXYCYCLINE HYDROCHLORIDE, 2.% SODIUM HYPOCHLORITE, 17% ETHYLENEDIAMINE TETRAACETIC ACID AND 0.2% CHLORHEXIDINE GLUCONATE AGAINST CANDIDA ALBICANS AN IN VITRO STUDY 18. Thomas J, Crout RJ, Crout RM, et al. Antibiotic resistance evaluation with low dose doxycycline (LDD) in adult periodontitis. J Dent Res 199;74(Special Issue):7 (Abstr.1394). 19. Rahmat A, Barkhordar, Larry G et al. Removal of intracanal smear by doxycycline in vitro. Oral Surg Med Oral Pathol Oral Radiol Endod 1997;84: Ferrari PHP, Cai S, Bombana AC. Effect of endodontic procedures on enterococci, enteric bacteria and yeasts in primary endodontic infections. Int Endod J 200;38: Sen BH, Safavi KE, Spangberg LSW. Growth patterns of Candida albicans in relation to radicular dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1997;84: Bystrom A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89: Sen BH, Safavi KE, Spangberg LS. Colonisation of Candida albicans on cleaned human dental hard tissues. Arch Oral Biol 1997;42: Harrison JW, Wagner GW, Henry CA. Comparison of the antimicrobial effectiveness of regular and fresh scent Clorox. J Endod 1990;16: Sen BH, Safavi KE, Spångberg LS. Antifungal effects of sodium hypochlorite and chlorhexidine in root canals. J Endod 1999 Apr;2(4): Ates M, Akdeniz BG, Sen BH. The effect of calcium chelating or binding agents on Candida albicans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 200 Nov;100(): Chambers HF. Antimicrobial agents: protein synthesis inhibitors and miscellaneous antibacterial agents. In: Hardman JG. Limbird LE, eds. Goodman & Gilman s The pharmacological basis of therapeutics, 10 th ed. New York: The McGraw-Hill Co., 2001; Ferguson JW, Hatton JF, Gillespie MJ. Effectiveness of intracanal irrigants and medications against the yeast Candida albicans. J Endod 2002 Feb;28(2): Siqueira Jr JF, Uzeda M, Fonseca MAF, A scanning electron microscopic evaluation of in vitro dentinal tubules penetration by selected anaerobic bacteria. J Endod 1996;22: Valera MC, de Moraes Rego J, Jorge AO. Effect of sodium hypochlorite and five intracanal medications on Candida albicans in root canals. J Endod 2001 Jun;27(6): The editorial board welcomes donation or advertisements Back Outer Cover Rs. 1,000/- Front Inner Cover Rs. 10,000/- Back Inner Cover Rs. 10,000/- Full Page Colour Rs.,000/- Full Page B/W Rs. 3,000/- The amount is payable at Mangalore by D.D. / Cheque / Pay Order in favour of Endodontology. The out-station Cheques should include Rs. 0/- 13