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Comparative study of effectiveness of disinfectants on Candida albicans.

INTRODUCTION: A considerable proportion of the population have detectable numbers of yeast in the mouth, yet very few of those people suffer from oral Candida infection. Placement of removable dentures in the oral cavity produces profound changes of the oral environment that may have an adverse effect on the integrity of the oral tissues, which might further change the oral microbial flora. Improper oral hygiene and negligence on denture care lead to candiasis (1). The denture cleansers currently available in the market are costly and are not readily available. The current study attempted at evaluating the effectiveness of house hold denture cleansers on Candida albicans (2).

MATERIAL AND METHODS: The present study was conducted in the Dept of Microbiology, Kurnool Medical College, Kurnool during 2013-2014.

An in vitro study was conducted to evaluate and compare the effectiveness of various household denture cleanser solutions aganist candida albicans.

STEPS FOLLOWED IN THE STUDY:

1. FABRICATION OF MASTER DIE FOR PREPARATION OF STUDY MOULD: For the study a precise, metal mould was custom fabricated with measurement of 10x10x2 mm, and was used to make test samples.

2. PREPARATION OF DENTAL PLASTER MOULD SPACE: Wax patterns were obtained by pouring wax into the die space present within the metal mould. The wax patterns obtained were checked for voids and inaccuracies. The wax patterns were then invested in a dental flask, using dental plaster following the manufacturer's instructions for water-powder ratio, mixing time and setting time. Mechanical vibrator was used to prevent air trapping during investing. Once the plaster was completely set, dewaxing was done for 10 min. The mould space thus obtained was used for the preparation of the test samples.

3. PREPERATION OF TEST SAMPLES: The denture base resins used were in ratio of 1:3 monomer and polymer for all the groups. Separating medium was applied on to the dental plaster mould with the help of a camel hair brush and dried. The material was mixed following the manufacturers' instructions and packed at dough stage. The specimens were bench cured for 1 hr and polymerized by short curing cycle according to manufactures instructions. After processing, the specimens were bench cooled for 1 hr and then finished with various grits of sand paper, followed by polishing with pumice. Thickness, length and width of each specimen were verified. The specimens were stored in distilled water for 24 hrs.

GROUPING OF STUDY SPECIMENS:
SL.   GROUP                   COMPONENT                        NO. OF
NO                                                             SAMPLES

1     A Denture Cleanser-1    Heat polymerized acrylic resin     10
      (hydrogen peroxide)     specimens immersed in denture
                              cleanser solution
                              hydrogenperoxide for 8hrs.

2     B Denture Cleanser-2    Heat polymerized acrylic resin     10
      (alcohol, citricacid,   specimens immersed in denture
      bakingsoda)             cleanser solution (alcohol,
                              citricacid, bakingsoda) for
                              8hrs

3     C Denture Cleanser-3    Heat polymerized acrylic resin     10
      (household bleach)      specimens immersed in denture
                              cleanser solution of household
                              bleach 30 min

4     Denture Cleanser-4      Heat polymerized acrylic resin     10
      (10%vinegar)            specimens immersed in
                              distilled water for 24 hours.

5     Control Group           Heat polymerized acrylic resin     10
      (distilled water)       specimens immersed in
                              distilled water for 24 hours.


STERILIZATION OF ACRYLIC RESIN SPECIMEN: The acrylic resin specimens will be sterilized in ultraviolet light chamber for 5 minutes. As ultraviolet light does not penetrate opaque materials, the specimens will be overturned using sterile forceps and the other side will be sterilized for 5 minutes. The acrylic specimens will be then placed in sterilization pouches.

BROTH PREPARATION: Two to three colonies of Candida albicans grown on blood agar will be taken and incubated onto sabourards dextrose broth. This will be incubated aerobically at 37[degrees]C for 24 hours and should be matched to mcfarlands stand and unit 1.

[FIGURE 1 OMITTED]

PREPARATION OF DENTURE CLEANERS:

* 20ml of 3% [H.sub.2][O.sub.2]

* 10ml Alcohol+1teaspoon fullsoda+10ml citric acid.

* 1 teaspoon full washing soda+20ml distilled water.

* 20 ml 10%vinegar.

* 20ml normal tap water.(for control group).

EXPOSURE OF SPECIMENS TO CANDIDA ALBICANS: The sterile acrylic resin specimens of various groups will be immersed, with labelled surface facing down, in Petri dishes containing 20ml of Sabouraud dextrose broth containing Candida albicans. These will be incubated for 16 hours at 37[degrees]C in incubator. This will simulate the duration of denture worn by the patient during daytime. Then the inoculated specimens will be washed under running tap water to simulate the patient's routine denture cleaning procedure.

[FIGURE 2 OMITTED]

CANDIDA REMOVAL TEST: The specimens will be immersed in Petri dishes containing denture cleaning agents. They will be stored for 8 hours at room temperature. This mimics the overnight soaking of dentures in cleaners according to the manufacturer's instructions. After this the specimens will be washed under running tap water, will be fixed with methanol, stained with crystal violet, dried and will be examined under microscope.

COUNTING OF CANDIDA CELLS: Candida cells adherent to acrylic resin specimens will be counted under the microscope(x40 magnification). The number of cells adherent on the test samples will be compared with that adherent to control.

[FIGURE 3 OMITTED]

STATISTICAL ANALYSIS: Data collected by experiments were computerized and analyzed using the Statistical Package for Social Sciences (SPSS) version 15.0. Results were expressed in frequencies. Nonparametric tests namely ANOVA test and tukey test were used for testing the statistical significance. For all tests a p-value of 0.05 or less was considered for statistical significance.

RESULTS:
Figure 4
Figure: Comparison of five groups (H2O2, Alcohol, House Hold Bleach.
10% Vinegar and Water) with respect to CFU counts in before and after
immersion in denture cleanser

              CFU before immersion   CFU after immersion
              in denture cleanser    in denture cleanser

H2O2                 70.50                 50.00
Alcohol              83.20                 47.00
House Hold           79.50                 40.30
  Bleach             83.60                 48.40
10% Vinegar          90.10                 79.90
Water

Note: Table made from bar graph.


Table: Comparison of five groups ([H.sub.2][O.sub.2], Alcohol, House Hold Bleach, 10% Vinegar and Water) with respect to CFU counts in difference from before and after immersion in denture cleanser by one way ANOVA.
Sources of    Sum of     Degrees of   Mean sum     F-value    p-value
variation     squares     freedom     of squares

Between      6172.7200       4         1543.18     26.9065   0.00001 *
  groups
Within       2580.9000       45         57.35
  groups
Total        8753.6200       49

* p<0.05


DISCUSSION: Maintaining oral hygiene is essential even when some or all teeth have been replaced with removable dentures. A properly maintained prosthesis contributes to good oral health. With patient education and preventive dentistry, a dentist must be able to recommend a denture cleanser that is effective, shows less deleterious effects on denture base materials and compatible with oral tissues. (3)

The process by which dentures accumulate plaque, stain and calculus is apparently similar to that process which takes place on natural teeth. Tonzetich studied the causes of halitosis extensively and concluded that a program of good oral hygiene can effectively control odor in most instances of oral origin. Theliade suggested that the presence of denture plaque constitutes the principal cause leading to inflammation of the palatal mucosa.

According to Budtz-Jorgensen this inflammation in edentulous patients can best be prevented by meticulous oral and denture hygiene. (4)

An ideal denture cleanser should have bactericidal and fungicidal properties, be simple to use, effectively remove organic and inorganic matter from denture surfaces, and be compatible with all denture materials. (5, 6) Denture cleansers are widely used to prevent colonization by Candida albicans and related Candida species, and to prevent denture plaque formation. (7, 8)

Chemical cleansing systems can be divided into 5 different groups, depending on their chemical constituents and mechanism of action. These include alkalineperoxides, alkaline hypochlorites, acids, disinfectants, and enzymes. Acrylic resin specimens of the size 10x10x2mm were fabricated using customized metal mould. A total of 50 standardized specimens were fabricated and stored in distilled water for 24hrs. After 24 hrs, the specimens were appropriately labelled and immersed in groups of 10 to prepared denture cleanser solutions for treatment. Solutions were prepared and immersed according to manufactures instructions. The removal of adherent cells from the denture base surface is an important part of denture cleansing. (9) The Candida removal test is a quantitative measurement of the efficacy of denture cleanser. (10) As such, in this study Candida removal test was performed to check the efficacy of denture cleaning agents. The present study evaluated and compared effectiveness of different denture cleansers in removing Candida from the surface of acrylic resin specimens.

After preparation of specimens Two to three colonies of Candida albicans grown on blood agar will be taken and incubated onto sabourards dextrose broth. This will be incubated aerobically at 37[degrees]c for 24 hours. Candidal growth is seen in sabourards dextrose broth and is matched to mcfarlands standard 1 which is equal to 1 x10-6 candidal cells per millilitre. Exposure of specimens to Candida albicans present in broth the sterile acrylic resin specimens of various groups by immersing in to broth with labelled surface facing down, in Petridishes containing 20ml of Sabouraud dextrose broth containing Candida albicans. These will be incubated for 16 hours at 37[degrees]c in incubator. This will simulate the duration of denture worn by the patient during daytime. Then the inoculated specimens will be washed under running tap water to simulate the patient's routine denture cleaning procedure. Then candidal colony count of each specimen is calculated and noted as initial colony count of 50 specimens. After that Candida removal test is done by immersing the specimens in Petridishes containing denture cleaning agents. They will be stored for 8 hours at room temperature. This mimics the overnight soaking of dentures in cleaners according to the manufacturer's instructions. After this the specimens will be washed under running tap water, will be fixed with methanol, stained with crystal violet, dried and will be examined under microscope. Candida cells adherent to acrylic resin specimens will be counted under the microscope(x40 magnification). The number of cells adherent on the test samples will be compared with that adherent to control and the initial count obtained while immersion into the sabrouds dextrose broth. The findings for all the groups were tabulated and statistically analyzed using POST HOC TUKEY TEST, ONE WAYANOVA TEST,PARIED "T" TEST was done. The 'P' value of <0.05 was considered significant. As seen from table 1 and graph 1, group the control (n=10) consisting of denture base acrylic specimens immersed in distilled water (10x10x2 mm), The mean colony count of candida on denture base acrylic specimens after immersion in Group A (hydrogen peroxide denture cleansers) ranged from a maximum of 70.50 before immersion, to a minimum of 50 after immersion into denture cleanser,. The mean colony count of candida on denture base acrylic specimens after immersion in Group b (alcohol, bakingsoda, citricacid combination denture cleansers) ranged from a maximum of 83.20 before immersion, to a minimum of 47 after immersion into denture cleanser. The mean colony count of candida on denture base acrylic specimens after immersion in Group c (household bleach denture cleansers) ranged from a maximum of 79.50 before immersion, to a minimum of 40.30 after immersion into denture cleanser. The mean colony count of candida on denture base acrylic specimens after immersion in Group d (vinegar denture cleansers) ranged from a maximum of 83.60 before immersion, to a minimum of 48.40 after immersion into denture cleanser. The mean colony count of candida on denture base acrylic specimens after immersion in Group e (water *control) ranged from a maximum of 90.10 before immersion, to a minimum of 79.90 after immersion into denture cleanser. As seen fromTable:2 Comparison of five groups ([H.sub.2][O.sub.2], Alcohol, House Hold Bleach, 10% Vinegar and Water) with respect to CFU counts in before immersion in denture cleanser by one way ANOVA between groups and within groups, the "p" value is 0.01 which is less than 0.05 which is statistically significant. As seen from the Table:3 Pair wise comparison of five groups ([H.sub.2][O.sub.2], Alcohol, House Hold Bleach, 10% Vinegar and Water) with respect to CFU counts before immersion in denture cleanser by Tukeys multiple post hoc procedures the "p" value is less than 0.05 when compared pair wise between groups, so stastically significant.

CONCLUSION:

Within the limitations of the study, the following conclusions were drawn:

The colony count decreases after immersion in to household denture cleanser solutions like baking soda, Sodium hypochlorite, alcohol, vinegar, household bleach, and were clinically significant.

House hold bleach showed more reduction in colony count of candida albicans followed by alcohol +baking soda+lemon juice combination, vinegar, [H.sub.2][O.sub.2] and water respectively.

DOI: 10.14260/jemds/2015/93

ACKNOWLEDGEMENT: Authors are thankful to Department of Microbiology, KMC, Kurnool.

REFERENCES:

(1.) Falah-Tafti,. A. jafari and M.H. Lotfi-Kamran, Comparison of the Effectiveness of Sodium Hypochlorite and Dentamize Tablet for Denture Disinfection World J. Med. Sci., 3 (1): 10-14, 2008 Renata C, Garcia R, Leon BL, Oliveira VMB, Cury A. Effect of denture cleanser on weight, surface roughness and tensile bond strength of two denture liners. J Prosthet Dent 2003; 89: 489-94.

(2.) Peracini A, Davi L R, Ribeiro N, De Souza RF, Silva CHL, Paranhos HFO. Effect of denture cleansers on physical properties of heat polymerized acrylic resin. J Prosthodont Res 2010; 54: 78-83.

(3.) Sheen R.S, Harrison A. Assessment of plaque prevention on dentures using an experimental cleanser. J Prosthet Dent 2000; 84: 594-601.

(4.) Dr. Chethan M D, Dr. N. S. Azhagarasan, Dr. Saketmiglani, Dr. Mohammed. H. S, Dr. A. Hari Prasad microbiological evaluation of the effectiveness of commercially available denture cleansing agents International Journal of Drug Development & Research July-September 2011 | Vol. 3 | Issue 3| ISSN 0975-9344.

(5.) Nandithakumar.et.al Efficacy of commercial and house hold denture cleansers against candida albicans adherent to acrylic denture base resin:an in vitro study.indianjournal of dental research 2012: 23 (1): 39-42.

(6.) Microbiological evaluation of the effectiveness of commercially available denture cleansing agents int.j.drug dev. & res., 2011, 3 (3): 159-172.

(7.) Sanitization of dentures by several denture hygiene methods jpd 1984(52):158-163. Marina Xavier Pisani, Claudia Helena Lovato da Silva, Helena de Freitas Oliveira Paranhos, Raphael Freitas Souza, Ana Paula Macedo The Effect of Experimental Denture Cleanser Solution Ricinuscommunis on Acrylic Resin Properties Materials Research. 2010;13(3):369-373.

(8.) Calcus and stain removal from acrylic resin dentures jpd 1968 (20): 326-329 Z. Harrison, a. Johnson & c. W. I. Douglas An in vitro study into the effect of a limited range of denture cleaners on surface roughness and removal of Candida albicans from conventional heat-cured acrylic resin denture base material, Journal of Oral Rehabilitation 2004 31; 460-467.

(9.) A comparative study to determine the commercially available denture cleansers. ijds. 2013 (5) 1-5.

(10.) Frederico Silva de Fernandes, Tatiana PereiraCeni, Wander Jose da Silva, Antonio Pedro Ricomini Filho, Fabiana Gouveia Straioto and Altair Antoninha Del Bel Cury. Efficacy of denture cleaners on Candida Spp. biofilm formed on polyamide and polymethyl methacrylate resins. J Prosthet Dent 2010; 105: 51-58.

B. Naga Jyothi [1], P. Venkateswarlu [2], B. Shanti Reddy [3], S. Umadevi [4]

AUTHORS:

[1.] B. Naga Jyothi

[2.] P. Venkateswarlu

[3.] B. Shanti Reddy

[4.] S. Umadevi

PARTICULARS OF CONTRIBUTORS:

[1.] Associate Professor, Department of Microbiology, Government Medical College, Anantapuramu.

[2.] Associate Professor, Department of Microbiology, Government Medical College, Anantapuramu.

[3.] Associate Professor, Department of Microbiology, Government Medical College, Anantapuramu.

[4.] Assistant Professor, Department of Microbiology, Government Medical College, Anantapuramu.

NAME ADDRESS EMAIL ID OF THE CORRESPONDING AUTHOR:

Dr. B. Naga Jyothi, Associate Professor, Department of Microbiology, Government Medical College, Anantapuramu.

E-mail: nagajyothibeldonu@gmail.com

Date of Submission: 05/01/2015.

Date of Peer Review: 06/01/2015.

Date of Acceptance: 07/01/2015.

Date of Publishing: 09/01/2015.
Figure 5
Figure: Comparison of % of reduction of CFU counts from
before and after immersion in denture cleanser in five groups

              % of reduction

House Hold        49.3
  Bleach
Alcohol           43.5
10% Vinegar       42.1
H2O2              29.1
Water             11.3

Note: Table made from bar graph.
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Title Annotation:ORIGINAL ARTICLE
Author:Jyothi, B. Naga; Venkateswarlu, P.; Reddy, B. Shanti; Umadevi, S.
Publication:Journal of Evolution of Medical and Dental Sciences
Article Type:Report
Date:Jan 12, 2015
Words:2684
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