Effectiveness of a 2% Chlorhexidine Solution Mixed With Calcium

8/22/2019 Effectiveness of a 2% Chlorhexidine Solution Mixed With Calcium

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T E C H N I C A L N O T E

Effectiveness of a 2% chlorhexidine solution mixed with calciumhydroxide against Candida albicansSaad Al-Nazhan, BDS, MSD and Mohammed Al-Obaida, DDS, MSEd, FRCD(C)

Division of Endodontics, Department of Restorative Dental Science, College of Dentistry, King Saud University, Riyadh, Saudi Arabia

Keywords

calcium hydroxide, Candida albicans,

chlorhexidine.

Correspondence

Professor Saad Al-Nazhan, King Saud

University, College of Dentistry, Department of

Restorative Dental Science, Division of

Endodontics, P.O. Box 60169 Riyadh, Saudi

Arabia 11545. Email: [email protected]

doi:10.1111/j.1747-4477.2007.00091.x

Abstract

The purpose of this investigation was to examine the in vitro susceptibility of

Candida albicans to a mixture of chlorhexidine and calcium hydroxide using a

tube dilution test. A suspension containing C. albicans was exposed to 2%

chlorhexidine solution, calcium hydroxide paste and 2% chlorhexidine mixed

with calcium hydroxide in plastic tissue culture wells. The tested agents were

incubated with C. albicans for 1, 24, or 72 h. All agents were effective anti-

fungals at all experimental time periods tested, with the exception of exposure

to calcium hydroxide for 1 h. A mixture of 2% chlorhexidine and calcium

hydroxide therefore is a very effective anti-fungal agent against C. albicans.

Introduction

The presence of Candida albicans in the infected pulp and

periradicular areas has been demonstrated through the

use of light and electron microscopy, as well as using

culture techniques (14). The incidence of C. albicans ininfected root canals has been shown to vary between 7%

and 55% (5,6) and occurs in approximately 11.36% of

teeth with pulp lesions (7). It has also been reported that

C. albicans can use dentin as a nutrient source (8).

Several studies have tested the effect of intracanal

irrigants and antiseptic agents on C. albicans, including

sodium hypochlorite, chlorhexidine solution and

calcium hydroxide paste (911). These antiseptic agents

have been shown to be very effective against C. albicans.

Waltimo et al. (4) evaluated different oral Candida spp.,

including C. albicans, in the presence calcium hydroxide

in vitro. They reported that Candida spp. were resistant to

calcium hydroxide and concluded that there is a need

for supplementary agents to effectively treat persistent

apical periodontitis.

Different vehicles have been added to calcium hydrox-

ide in an attempt to enhance its antimicrobial activity.

Estrela et al. (12) tested the anti-fungal effectiveness of

different mixtures of calcium hydroxide using saline,

polyethylene glycol and camphorated paramonochlo-

rophenol. They reported complete anti-fungal effective-

ness after 1 h of exposure, irrespective of the vehicle

associated with the calcium hydroxide paste.

Chlorhexidine has been suggested to be a useful endo-

dontic irrigant and intracanal antiseptic due to its low

toxicity and excellent anti-bacterial and anti-fungal

activities (11,13). A combination of chlorhexidine andcalcium hydroxide was tested against bacterial species

only, and was reported to be effective against Enterococcus

faecalis (14,15). It would therefore be of value to deter-

mine the effectiveness of this combination of agents

against C. albicans.

The purpose of the current study was to examine the

in vitro susceptibility of C. albicans to a mixture of chlor-

hexidine and calcium hydroxide.

Materials and methods

The anti-fungal effect of 2% chlorhexidine solution,

calcium hydroxide paste and 2% chlorhexidine mixed

with calcium hydroxide was evaluated against C. albicans.

Stock cultures of clinical isolates of C. albicans were

provided by the Microbiology Laboratory of King Khalid

University Hospital (Riyadh, Saudi Arabia) and main-

tained in Sabourauds dextrose agar plates.

A suspension was prepared by transferring three colo-

nies from a Sabourauds dextrose agar plate, using a

sterile 4 mm diameter platinum loop, to a 10 mm

Aust Endod J 2008; 34: 133135

The Journal of the Australian Society of Endod ontology Inc. andthe Australian and New Zealand Academy of Endodontists

133 2007 The Authors

Journal compilation 2007 Australian Society of Endodontology
mailto:[email protected]:[email protected]


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Sabouraud infusion broth in a sterilised 10 mL screw

capped test tube, followed by incubation for 1 week at

37oC. Two such test tubes were prepared.

Experimental procedure

The experiments were performed in plastic tissue cultureclusters containing 24 wells, each with an inner diameter

of 16 mm. One mL of test material was placed at the

bottom of each culture well, to which 1 mL of a Candida

suspension was added. At the same time, 1 mL of Sab-

ouraud infusion broth media was mixed with 1 mL of a

Candida suspension in a culture well to serve as a positive

control. For the negative control, 2 mL of Sabouraud

infusion broth was placed in a culture well. Six wells were

thus used per test. The culture clusters plates were then

incubated at 37oC and evaluated after 1, 24 and 72 h.

At the end of the incubation period, aliquots of 0.1 mL

were withdrawn from each well and transferred to tubes

containing 5 mL of fresh Sabouraud infusion broth. The

tubes were then vortexed, incubated at 37oC, and

observed for 7 days. Growth of the fungi was observed

daily, as indicated by the presence of turbidity in the

tubes. The presence of turbidity was determined, and the

purity of the cultures was checked by the morphology of

colonies onto blood agar plates. The results were statisti-

cally analyzed using the KruskalWallis test.

Results

The positive controls, in which there was no putative

anti-fungal agent, exhibited growth of C. albicans at alltimes tested; as expected, no growth occurred in the

negative control group. Calcium hydroxide was ineffec-

tive at inhibiting the growth of C. albicans after 1 h of

exposure. Further, there was no significant difference

(P> 0.05) in growth between calcium hydroxide treat-

ment for 1 h and the growth in the positive control.

The exposure of C. albicans to calcium hydroxide at 24

and 72 h, however, resulted in complete inhibition of

growth. The growth of C. albicans was completely inhib-

ited when exposed to 2% chlorhexidine alone or when

mixed with calcium hydroxide at all times tested. Statis-

tical analysis showed no significant difference between

growth of C. albicans exposed to 2% chlorhexidine alone

or mixed with calcium hydroxide and the negative

control (P> 0.05).

Discussion

The method used to evaluate inhibition of fungal growth

in this study was the dilution tube susceptibility test,

which is an effective method to evaluate anti-fungal as

well as anti-bacterial properties of any filling material or

solution (16,17). This method allows direct contact in the

solution between C. albicans and the agent to be tested.

The growth of C. albicans observed in the positive controls

confirmed the efficiency of the methodology. Two agents,

chlorhexidine and calcium hydroxide, were chosen for

this study. Chlorhexidine is a broad-spectrum anti-microbial agent that has been reported to be an effective

antiseptic in endodontic therapy owing to its unique

ability to bind to dentin and its substantivity in the root

canal system. It is also effective against strains resistant to

calcium hydroxide (18,19).

In a study comparing common endodontic disinfec-

tants, 0.5% chlorhexidine was also significantly more

effective at killing C. albicans than calcium hydroxide, 5%

and 0.5% sodium hypochlorite and 2% iodine potassium

iodide (4). In the present study, a high concentration of

chlorhexidine (2%) was used and was found to be effec-

tive against C. albicans at all experimental times tested.

Similar findings were reported by Sen et al. (11), who

found that a 1 h exposure to a concentration of 1200 mg

of chlorhexidine/mL was sufficient to eliminate C. albicans

from dentinal surfaces with or without the presence of a

smear layer.

Recent studies have questioned the efficacy of calcium

hydroxide in reducing microbial numbers, even after pro-

longed contact with the root canal (20). Waltimo et al. (4)

reported that C. albicans survived incubation in calcium

hydroxide solution for 1 h and was killed after 6 h of

incubation. Similar findings were obtained in the current

study. Supplementing calcium hydroxide with chlorhexi-

dine may therefore be one way to improve the efficacy ofits anti-fungal activity, while also maximising microbial

eradication when used as an intracanal dressing. Chlo-

rhexidine, in different concentrations and in combination

with calcium hydroxide, maintain its physicochemical

properties (i.e. pH, radio-opacity and working time) that

allow it to be used as an intracanal antiseptic agent

(21,22). In the present investigation, mixing calcium

hydroxide with chlorhexidine was found to be very effec-

tive against C. albicans at all experimental times tested.

The anti-bacterial property of calcium hydroxide depends

on its long-lasting alkalinity and has been reported not to

be affected by the addition of chlorhexidine (15). Such a

combination will result in better diffusion into the den-

tinal tubules of prepared root canal systems with the

potential to be used as a long-term intracanal agent and

therefore complete anti-microbial, as well as anti-fungal,

effectiveness can be improved.

In conclusion, the results of the present study show

the additive benefits achieved by combining calcium

hydroxide with chlorhexidine in the treatment of

C. albicans.

Chlorhexidine, Calcium Hydroxide, Candida albicans S. Al-Nazhan and M. Al-Obaida




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S. Al-Nazhan and M. Al-Obaida Chlorhexidine, Calcium Hydroxide, Candida albicans



Effectiveness of a 2% Chlorhexidine Solution Mixed With Calcium

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